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EMS Mutagenesis of Clostridium difficile to Identify Strains with Germination-null Phenotypes
EMS诱变鉴定艰难梭菌萌发无效显型菌株   

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Abstract

Clostridium difficile is a Gram-positive, spore-forming, strict anaerobe and the leading cause of antibiotic-associated diarrhea (McFarland, 2008). Germination by C. difficile spores is the first step in pathogenesis. Thus, identifying mechanisms of C. difficile spore germination may lead to novel anti-germination therapies. This protocol describes a method for identifying germination-null phenotypes for C. difficile spores by introducing random mutations into actively growing C. difficile using ethyl methanesulfonate (EMS) as a mutagen (Francis et al., 2013).

Keywords: Clostridium difficile(难辨梭状芽孢杆菌), Germination(发芽), Spore(孢子), Mutagenesis(诱变)

Materials and Reagents

  1. A petri plate of viable C. difficile cells
  2. Ethyl methanesulfonate (EMS)
  3. Rifampin
  4. Sterile water
  5. Sucrose
  6. 10% (w/v) taurocholic acid
  7. Thioglycollate
  8. Lysozyme
  9. Brain Heart Infusion (BD BactoTM, catalog number: 237500 )
  10. Yeast Extract (BD DifcoTM, catalog number: 212750 )
  11. 0.1% (w/v) L-cysteine (Sigma-Aldrich, catalog number: C7352 )
  12. BHIS medium (see Recipes)
  13. BHIS agar medium (see Recipes)

Equipment

  1. 15 ml conical screw cap tubes
  2. 50 ml conical screw cap tubes
  3. Anaerobic chamber
  4. Spectrophotometer or microplate reader
  5. Biosafety cabinet
  6. Bench-top microcentrifuge
  7. Swinging-bucket centrifuge
  8. 65 °C heat block
  9. 50 °C water bath
  10. 37 °C water bath

Procedure

  1. Mutagenesis
    1. In the anaerobic chamber, inoculate 5 ml BHIS medium with a colony of C. difficile and grow overnight at 37 °C.
      Note: The use of 15 ml or 50 ml conical screw cap tubes will facilitate washing steps and incubation steps. However, they are not absolutely required for the protocol to work.
    2. The next day, back dilute the overnight culture into 5-ml BHIS medium to OD600 = 0.05 and grow at 37 °C to an OD600 = 0.5.
      Note: OD600 = 0.5 is mid-log phase growth. A log phase culture is necessary to ensure that the introduced mutations are passed efficiently to each generation.
    3. Add EMS to 1% (v/v) and grow for 3 h.
      Notes:
      1. Be sure to properly suspend the EMS, it can form globules at the bottom of the tube.
      2. EMS is a powerful DNA mutagen. Dispose of waste in accordance with your institutions protocols.
    4. Wash the culture, twice, by centrifuging for 10 min at 3,000 x g and at room temperature with 10 ml BHIS medium to remove the EMS.
    5. Resuspend the pellet in 40 ml BHIS medium.
    6. Recover the cells overnight in the BHIS medium at 37 °C.
      Note: The frequency of rifampin resistance in this population will help determine if the mutagenesis of the cells was successful. Typically, an approximately 1,000 x increase in rifampin resistance is observed.

  2. Isolation of germination-null phenotypes
    1. Spread 50 μl of the recovered, mutagenized C. difficile cells onto each of 20 BHIS agar petri plates.
      Note: C. difficile sporulates most efficiently on agar medium. Addition of more cells to the plate results in fewer spores generated. We have found that a lower inoculum (e.g. 50 μl) results in a larger spore yield.
    2. Incubate plates at 37 °C in the anaerobic chamber until spores are formed (approximately 4-7 days, depending on the C. difficile strain used).
    3. Using a sterile loop or a pipette tip, scrape the growth from the plate and suspend the growth from each plate in a 1.5-ml microcentrifuge tube containing 1 ml of sterile water/plate.
      Note: All C. difficile manipulations outside of the anaerobic chamber should be done in accordance with your institutions IBC (e.g. in a biosafety cabinet).
    4. Incubate overnight at 4 °C without agitation.
      Note: Overnight incubation at 4 °C in water aids in the release of spores from the mother cell.
    5. Wash with 1-ml sterile water five times by centrifuging for 1 min at 14,000 x g in a microcentrifuge at room temperature.
      Note: As the vegetative cells/mother cells lyse, a white layer of cell debris is often seen. This layer appears on top of the spore layer and can be safely removed during washes.
    6. Combine the spore pellets into 3 ml water.
    7. Carefully layer the spore suspension on top of 7 ml 50% sucrose.
    8. Centrifuge for 20 min at room temperature and 4,000 x g.
    9. Remove the water layer and sucrose from the tube.
      Note: Spores form a pellet at the bottom of the tube. Vegetative cells and cell debris remain at the interface or dispersed within the sucrose fraction.
    10. Wash the purified spores, as described above in step B5, five times in sterile water.
    11. Suspend the purified spores in 40 ml BHIS medium supplemented with 10% (w/v) taurocholic acid.
      Notes:
      1. Taurocholic acid is a germinant for C. difficile spores.
      2. Filter sterilize the BHIS + 10% taurocholic acid medium before use.
    12. Incubate overnight at 37 °C with gentle agitation (e.g. rocking).
      Note: This condition will stimulate the germination of those spores that are still able to respond to taurocholic acid as a germinant. Because this protocol selections for germination-null phenotypes, the conditions at which the spores are germinated (i.e. aerobic vs. anaerobic) do not matter.
    13. Centrifuge the solution at room temperature for 10 min at 3,000 x g to pellet the spores and germinated spores.
    14. Resuspend the pellet in 10 ml BHIS medium and incubate aerobically at 65 °C for 60 min.
      Note: Only dormant spores will survive this condition.
    15. Centrifuge the solution as step B13.
    16. Resuspend the pellet in 250 mM thioglycollate.
      Note: Because this method selects for germination-null phenotypes, spores must be artificially germinated to recover the mutants as colonies (Kamiya et al., 1989).
    17. Incubate for 30 min at 50 °C.
    18. Centrifuge the solution as step B13.
    19. Resuspend the pellet in water supplemented with 4 mg/ml lysozyme.
    20. Incubate for 15 min at 37 °C.
    21. Spread 25 μl aliquots on 20 BHIS agar plates.
      Note: To enrich for germination-null phenotypes, repeat steps B1–B21.

Recipes

  1. BHIS Medium
    37 g/L Brain Heart Infusion
    5 g/L Yeast Extract
    0.1% (w/v) L-cysteine
  2. BHIS agar medium
    BHIS medium
    1.5% (w/v) agar

Acknowledgments

We would like to thank Charlotte Allen and Ritu Shrestha for aiding in the development of this protocol. This work was supported by the American Heart Association National Scientist Development grant to JAS (No. 11SDG7160013). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

  1. Francis, M. B., Allen, C. A., Shrestha, R. and Sorg, J. A. (2013). Bile acid recognition by the Clostridium difficile germinant receptor, CspC, is important for establishing infection. PLoS Pathog 9(5): e1003356.
  2. Kamiya, S., Yamakawa, K., Ogura, H. and Nakamura, S. (1989). Recovery of spores of Clostridium difficile altered by heat or alkali. J Med Microbiol 28(3): 217-221.
  3. McFarland, L. V. (2008). Update on the changing epidemiology of Clostridium difficile-associated disease. Nat Clin Pract Gastroenterol Hepatol 5(1): 40-48.

简介

艰难梭菌是革兰氏阳性,孢子形成,严格厌氧菌和抗生素相关性腹泻的主要原因(McFarland,2008)。 通过C发芽。 艰难梭菌孢子是发病机理的第一步。 因此,识别C的机制。 艰难梭菌孢子萌发可能导致新的抗萌发治疗。 该协议描述了鉴定em的萌发 - 无效表型的方法。 艰难梭菌孢子通过将随机突变引入活跃生长的C中来实现。 使用甲磺酸乙酯(EMS)作为诱变剂(Francis et al。,2013)来检测艰难梭菌。

关键字:难辨梭状芽孢杆菌, 发芽, 孢子, 诱变

材料和试剂

  1. 活的培养皿。 艰难细胞细胞
  2. 甲磺酸乙酯(EMS)
  3. 利福平
  4. 无菌水
  5. 蔗糖
  6. 10%(w/v)牛磺胆酸
  7. 硫代乙醇酸盐
  8. 溶菌酶
  9. 脑心浸液(BD Bacto TM ,目录号:237500)
  10. 酵母提取物(BD Difco TM ,目录号:212750)
  11. 0.1%(w/v)L-半胱氨酸(Sigma-Aldrich,目录号:C7352)
  12. BHIS介质(参见配方)
  13. BHIS琼脂培养基(见配方)

设备

  1. 15 ml锥形螺帽管
  2. 50ml锥形螺帽管
  3. 厌氧室
  4. 分光光度计或酶标仪
  5. 生物安全柜
  6. 台式微量离心机
  7. 摆动离心机
  8. 65°C热块
  9. 50°C水浴
  10. 37°C水浴

程序

  1. 诱变
    1. 在厌氧室中,接种5ml具有C的菌落的BHIS培养基。 艰难梭菌并在37℃下生长过夜 注意:使用15 ml或50 ml锥形螺旋盖管将有助于清洗步骤和孵育步骤。 但是,协议并不是绝对必需的。
    2. 第二天,将过夜培养物稀释到5-ml BHIS培养基中至OD 600 = 0.05,并在37℃下生长至OD 600 = 0.5。
      注意:OD 600 = 0.5是对数中期增长。 需要对数期培养以确保引入的突变有效地传递给每一代。
    3. 将EMS加入1%(v/v)并生长3小时 注意:
      1. 确保正确挂起EMS,它可以在管底部形成小球。
      2. EMS是一种强大的DNA诱变剂。 按照您的机构协议处理废物。
    4. 洗涤培养物两次,在3,000×g离心10分钟,在室温下用10ml BHIS培养基离心以除去EMS。
    5. 将沉淀重悬于40ml BHIS培养基中
    6. 在37℃下在BHIS培养基中恢复细胞过夜 注意:在该群体中利福平抗性的频率将有助于确定细胞的诱变是否成功。通常,观察到利福平耐药性增加约1,000倍。

  2. 发芽无效表型的分离
    1. 传播50微升的回收,诱变的。艰难细胞接种到20个BHIS琼脂培养皿上 注意:艰难梭菌在琼脂培养基上最有效地产孢。向板中加入更多的细胞导致产生更少的孢子。我们已经发现,较低的接种物(例如50μl)导致较大的孢子产量。
    2. 在37℃下在厌氧室中孵育平板,直到形成孢子(大约4-7天,取决于使用的艰难梭菌菌株)。
    3. 使用无菌环或移液器吸头,从板上刮下生长物,并在含有1ml无菌水/板的1.5ml微量离心管中悬浮每个板的生长。
      注意:在厌氧室外的所有艰难梭菌操作应根据您的机构IBC(例如在生物安全柜中)进行。
    4. 在4℃下不搅拌孵育过夜 注意:在4℃下在水中过夜孵育有助于从母细胞释放孢子。
    5. 用微量离心机在室温下以14,000×g离心1分钟,用1ml无菌水洗涤5次。
      注意:当营养细胞/母细胞溶解时,经常看到白色的细胞碎片层。 该层出现在孢子层的顶部,并且可以在洗涤期间安全地移除。
    6. 将孢子颗粒合并到3ml水中。
    7. 小心地将孢子悬浮液铺在7ml 50%蔗糖的顶部
    8. 在室温和4000xg离心20分钟。
    9. 从管中除去水层和蔗糖。
      注意:孢子在试管底部形成沉淀。 营养细胞和细胞碎片保留在界面处或分散在蔗糖部分内。
    10. 如上述步骤B5中所述,将纯化的孢子在无菌水中洗涤五次
    11. 将纯化的孢子悬浮于补充有10%(w/v)牛磺胆酸的40ml BHIS培养基中 注意:
      1. 天门冬氨酸是艰难梭菌芽孢的发芽物。
      2. 在使用前过滤灭菌BHIS + 10%牛磺胆酸培养基。
    12. 在37℃下温和搅拌过夜(例如摇动)。
      注意:这种条件将刺激仍然能够对牛磺胆酸作为发芽物反应的那些孢子的萌发。 因为这个协议选择萌发无效的表型,孢子发芽的条件(即有氧与厌氧)无关紧要。
    13. 将溶液在室温下以3,000xg离心10分钟以沉淀孢子和发芽的孢子。
    14. 将沉淀重悬在10ml BHIS培养基中,在65℃下有氧培养60分钟 注意:只有休眠孢子能够在这种情况下存活。
    15. 离心溶液,如步骤B13
    16. 将沉淀重悬在250mM巯基乙酸盐中 注意:因为该方法选择萌发无效表型,所以必须人工发芽孢子以恢复作为集落的突变体(Kamiya等人,1989)。
    17. 在50℃下孵育30分钟。
    18. 离心溶液,如步骤B13
    19. 将沉淀重悬于补充有4mg/ml溶菌酶的水中
    20. 在37℃下孵育15分钟。
    21. 将25μl等分试样在20个BHIS琼脂平板上铺平 注意:要丰富萌发无效的表型,请重复步骤B1-B21。

食谱

  1. BHIS中
    37 g/L脑心脏输液
    5 g/L酵母提取物
    0.1%(w/v)L-半胱氨酸
  2. BHIS琼脂培养基
    BHIS介质
    1.5%(w/v)琼脂

致谢

我们要感谢Charlotte Allen和Ritu Shrestha帮助开发这个协议。这项工作是由美国心脏协会国家科学家发展赠款给JAS(No. 11SDG7160013)支持。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. Francis,M.B.,Allen,C.A.,Shrestha,R。和Sorg,J.A。(2013)。 由艰难梭菌发芽受体(CspC)识别胆汁酸是重要的用于建立感染。 PLoS Pathog 9(5):e1003356。
  2. Kamiya,S.,Yamakawa,K.,Ogura,H。和Nakamura,S。(1989)。 回收热或碱改变的艰难梭菌的孢子。 a> J Med Microbiol 28(3):217-221
  3. McFarland,L.V。(2008)。 关于艰难梭菌相关疾病的流行病学变化的更新。 Nat Clin Pract Gastroenterol Hepatol 5(1):40-48。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Francis, M. B. and Sorg, J. A. (2013). EMS Mutagenesis of Clostridium difficile to Identify Strains with Germination-null Phenotypes. Bio-protocol 3(24): e1001. DOI: 10.21769/BioProtoc.1001.
  2. Francis, M. B., Allen, C. A., Shrestha, R. and Sorg, J. A. (2013). Bile acid recognition by the Clostridium difficile germinant receptor, CspC, is important for establishing infection. PLoS Pathog 9(5): e1003356.
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