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Shigella uses a type III secretion system to invade host cell and to cause disease. Secretion control and insertion of a translocation pore into cell membrane are critical steps for pathogenesis and are tightly linked to the formation of the needle tip complex formed by the IpaB and IpaD proteins (Veenendaal et al., 2007). Surface localizations of IpaD and IpaB were monitored by FACS analysis according to the localization protocol for Pseudomonas aeruginosa homolog PcrV (Lee et al., 2010).

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Shigella IpaD and IpaB Surface Localizations
志贺氏杆菌IPaD和IPAB表面定位

微生物学 > 微生物生物化学 > 蛋白质 > 免疫检测
作者: Lionel Schiavolin
Lionel SchiavolinAffiliation: Molecular Bacteriology Lab, Université Libre de Bruxelles, Anderlecht, Belgium
For correspondence: lschiavo@ulb.ac.be
Bio-protocol author page: a992
Alaeddine Meghraoui
Alaeddine MeghraouiAffiliation: Molecular Bacteriology Lab, Université Libre de Bruxelles, Anderlecht, Belgium
Bio-protocol author page: a993
 and Abdelmounnaim Allaoui
Abdelmounnaim AllaouiAffiliation: Molecular Bacteriology Lab, Université Libre de Bruxelles, Anderlecht, Belgium
Bio-protocol author page: a994
Vol 3, Iss 22, 11/20/2013, 3502 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.975

[Abstract] Shigella uses a type III secretion system to invade host cell and to cause disease. Secretion control and insertion of a translocation pore into cell membrane are critical steps for pathogenesis and are tightly linked to the formation of the needle tip complex formed by the IpaB and IpaD proteins (Veenendaal et al., 2007). Surface localizations of IpaD and IpaB were monitored by FACS analysis according to the localization protocol for Pseudomonas aeruginosa homolog PcrV (Lee et al., 2010).
Keywords: Type 3 secretion system(3型分泌系统), Tip complex(尖端复杂的), Shigella(志贺菌), Flow cytometry(流式细胞仪)

[Abstract]

Materials and Reagents

  1. Shigella strains
  2. Tryptic Soy Broth (TSB) (VWR International, catalog number: for Europe 1.00525.5000 and for U.S. EM1.00525.5007 )
  3. Agar (MP Biomedicals, catalog number: 0 210026291 )
  4. Congo Red (VWR International, catalog number: for Europe 34140.184 )
  5. Anti-IpaD and -IpaB polyclonal antibodies (house made)
  6. PBS (Fischer Scientific, catalog number: BP399-20 )
  7. Paraformaldehyde (PFA) (Sigma-Aldrich, catalog number: P6148 )
  8. Triton X-100 (VWR International, for Europe catalog number: 1.08603.1000 )
  9. Trizmabase (Sigma-Aldrich, catalog number: T1503 )
  10. Bovine serum albumin (BSA) (VWR International, catalog number: 422361V )
  11. Anti-mouse secondary antibody CF647-conjugated (Sigma-Aldrich, catalog number: SAB4600351 )
  12. PBS + PFA 4% stock solution (see Recipes)
  13. Congo Red agar plates (see Recipes)

Equipment

  1. 250 ml Erlenmeyer
  2. Microtubes
  3. CR agar plate
  4. Polystyrene tube for FACS analysis
  5. Centrifuge Heating magnetic stirrer
  6. 37 °C shaker
  7. Rotator mixer
  8. Flow cytometry with a four-colour FACS Calibur cytometer (Becton Dickinson and Company)

Procedure

  1. Preparation of bacterial samples
    1. Launch overnight precultures in TSB (37 °C with shaking) from Congo Red (CR) positives colonies of Shigella on CR agar plates.
    2. Dilute 1: 100 precultures in fresh TSB (for volume see step A3) and incubate at 37 °C with shaking until an OD600 ≈ 1.5 is reached. (Medium must be filtered or autoclaved with stirring to avoid glucose caramelization which interferes with type III secretion.)
    3. Harvest 2 x 108 bacteria per tested conditions at 2,000 x g for 4 min at room temperature (RT) (OD600 of 1 correspond to approximately 5 x 108 bacteria).
    4. Wash twice with 500 μl of ice-cold PBS with 0.1% Triton X-100 (centrifugation conditions as in step A3). Be careful when you take out the liquid supernatant as the pellet can detach gradually all along the procedure.
    5. Resuspend in 500 μl of ice-cold PBS with 0.1% Triton X-100 and add 500 μl of PBS + PFA (4%) to fix bacteria.
    6. Mix by inversion and incubate 20 min at RT.
    7. Add 50 μl Tris HCl 1 M (pH 7.5), mix by inversion and incubate 5 min at RT to quench the cross-linker.
    8. Harvest bacteria at 10,000 x g for 2 min at RT (All further centrifugation steps are performed with these parameters.).
    9. Wash once with 1 ml PBS + 0.1% Triton X-100 and once with 1 ml PBS.

  2. Immunological staining
    1. Harvest bacteria and resuspend in 500 μl of the blocking solution (PBS + 4% BSA).
    2. Incubate bacteria on rotator mixer 1 h at 4 °C.
    3. Harvest bacteria and resuspend in 250 μl PBS + 4% BSA with mouse sera diluted 1: 100.
      Sera were from Swiss mice immunized with GST-IpaD131-332 (Schiavolin et al., 2013) or His-IpgC + IpaB (Page et al., 1999). Negative controls for IpaD and IpaB localizations are respectively an IpaD protein lacking its last residues which do not bind the needle tip (Espina et al., 2006) and an ipaD KO mutant where IpaB is not retained at the tip (Veenendaal et al., 2007). In both cases secreted IpaD or IpaB do not interfere with the assay.
    4. Incubate overnight at 4 °C with agitation.
    5. Add 750 μl PBS to bacteria and then wash twice with 1 ml PBS. All further steps are made in the dark to preserve fluorescent properties of the secondary antibody. Microtubes or rack are covered with aluminum foil.
    6. Resuspend in 250 μl of PBS + 4% BSA with goat CF647-conjugated anti-mouse IgG antibody diluted 1: 500.
    7. Agitate 1 h or longer at 4 °C (incubation time may last overnight).
    8. Add 750 μl PBS to bacteria and then wash twice with 1 ml PBS.
    9. Resuspend bacterial pellet in 500 μl of PBS and dilute 1: 10 in FACS polystyrene tube.
    10. Analyze by flow cytometry with a four-colour FACS Calibur cytometer for instance (you will find examples of results in the supporting information of the third reference (Schiavolin et al., 2013) available for free on Mol. Microbiol. website).
    11. Parameters used (no compensation):
      detectors
      voltage
      mode
      FSC
      E02
      Log
      SSC
      366
      Log
      FL4
      800
      Log

Recipes

  1. PBS + PFA 4% stock solution (100 ml)
    Weigh out 4 g of paraformaldehyde in a 250 ml erlenmeyer
    Add 80 ml of double-distilled water
    Add 50 μl of 1 M NaOH
    Add a magnetic bar and close the erlenmeyer
    Heat at 70 °C until complete solubilization (the solution should become clear)
    Put on ice and allow the solution to cool down to RT
    Adjust volume to 90 ml with double-distilled water
    Add 10 ml of PBS 10x and mix
    Filter solution with a 0.2 μm 25 mm nylon syringe filter and aliquot in 15 ml falcon tube
    Freshly prepared PFA can be stored at -20 °C for further assays (thaw gently at RT)
  2. Congo Red agar plates
    Prepare a 30 g/L TSB solution with bidistilled water and dissolve 15 g/L agar
    Autoclave the medium for 15 min at 120 °C (If you autoclave your medium for 20 min, sugar will caramelize and your plates will be darker, and even darkest after incubation with bacteria at 37 °C.)
    Add the Congo Red at a final concentration of 250 µg/ml (Stock solution is prepared in bidistilled water at a concentration of 10 mg/ml.) when the bottle can be safely handled with a protective glove.

Acknowledgments

This protocol was adapted from Lee et al. (2010). This study was supported by grants from the Belgian Fonds National de la Recherche Scientifique Médicale (FRS-FNRS; Convention 3.4556.11) and from the European Community’s Seventh framework program FP7/2011–2015 under grant agreement No. 261742. L.S. and A.M are recipients of a PhD fellowship from the Belgian Fonds National de Recherches Industrielles et Agronomiques (FRIA). A part of this work was also supported by the Fonds Defay, and by Van Buuren and Héger-Masson foundations. We thank A. Op de Beeck for her help in performing the FACS experiment.

References

  1. Espina, M., Olive, A. J., Kenjale, R., Moore, D. S., Ausar, S. F., Kaminski, R. W., Oaks, E. V., Middaugh, C. R., Picking, W. D. and Picking, W. L. (2006). IpaD localizes to the tip of the type III secretion system needle of Shigella flexneri. Infect Immun 74(8): 4391-4400.
  2. Lee, P. C., Stopford, C. M., Svenson, A. G. and Rietsch, A. (2010). Control of effector export by the Pseudomonas aeruginosa type III secretion proteins PcrG and PcrV. Mol Microbiol 75(4): 924-941. 
  3. Page, A. L., Ohayon, H., Sansonetti, P. J. and Parsot, C. (1999). The secreted IpaB and IpaC invasins and their cytoplasmic chaperone IpgC are required for intercellular dissemination of Shigella flexneri. Cell Microbiol 1(2): 183-193.
  4. Schiavolin, L., Meghraoui, A., Cherradi, Y., Biskri, L., Botteaux, A. and Allaoui, A. (2013). Functional insights into the Shigella type III needle tip IpaD in secretion control and cell contact. Mol Microbiol 88(2): 268-282.
  5. Veenendaal, A. K., Hodgkinson, J. L., Schwarzer, L., Stabat, D., Zenk, S. F. and Blocker, A. J. (2007). The type III secretion system needle tip complex mediates host cell sensing and translocon insertion. Mol Microbiol 63(6): 1719-1730.

材料和试剂

  1. 志贺氏菌菌株
  2. 胰蛋白酶大豆肉汤(TSB)(VWR国际,目录号:欧洲1.00525.5000和美国EM1.00525.5007)
  3. 琼脂(MP Biomedicals,目录号:0210026291)
  4. 刚果红(VWR国际,目录号:欧洲34140.184)
  5. 抗IpaD和-IpaB多克隆抗体(自制)
  6. PBS(Fischer Scientific,目录号:BP399-20)
  7. 多聚甲醛(PFA)(Sigma-Aldrich,目录号:P6148)
  8. Triton X-100(VWR International,欧洲目录号:1.08603.1000)
  9. Trizmabase(Sigma-Aldrich,目录号:T1503)
  10. 牛血清白蛋白(BSA)(VWR International,目录号:422361V)
  11. 抗小鼠第二抗体CF647缀合(Sigma-Aldrich,目录号:SAB4600351)
  12. PBS + PFA 4%储备液(见配方)
  13. 刚果红琼脂板(见配方)

设备

  1. 250 ml Erlenmeyer
  2. 微管
  3. CR琼脂平板
  4. 用于FACS分析的聚苯乙烯管
  5. 离心加热磁力搅拌器
  6. 37℃摇床
  7. 旋转搅拌器
  8. 使用四色FACS Calibur细胞计数器(Becton Dickinson and Company)的流式细胞术

程序

  1. 细菌样品的制备
    1. 在刚果红(CR)阳性的志贺氏菌菌落在CR琼脂平板上的TSB(37℃,摇动)中进行夜间预培养。
    2. 在新鲜TSB中稀释1:100预培养物(体积参见步骤A3),并在37℃下振荡温育直至达到OD 600 = 1.5。 (培养基必须过滤或高压灭菌,搅拌以避免葡萄糖焦糖化,干扰III型分泌)。
    3. 在室温(RT)下,在2,000×g下每个测试条件收获2×10 8个细菌4分钟(OD 600 = 1)对应于大约5×10 8个细菌)
    4. 用500μl含有0.1%Triton X-100的冰冷PBS洗涤两次(如步骤A3中的离心条件)。当取出液体上清液时要小心,因为颗粒可以沿着整个过程逐渐脱离
    5. 重悬在500μl冰冷的PBS与0.1%Triton X-100和添加500微升PBS + PFA(4%)以修复细菌。
    6. 通过倒置混合并在室温下孵育20分钟
    7. 加入50μlTris HCl 1M(pH 7.5),倒置混合,在室温下孵育5分钟以淬灭交联剂。
    8. 在RT下收获细菌(10,000×g)2分钟(用这些参数进行所有进一步的离心步骤)。
    9. 用1ml PBS + 0.1%Triton X-100洗涤一次,用1ml PBS洗涤一次
  2. 免疫染色
    1. 收获细菌,并在500μl封闭溶液(PBS + 4%BSA)中重悬
    2. 在旋转混合器上在4℃下孵育细菌1小时
    3. 收获细菌,并重悬在250μlPBS + 4%BSA中,用1:100稀释的小鼠血清 血清来自用GST-1paD 131-332(Schiavolin等人,2013)或His-IpgC + IpaB免疫的Swiss小鼠(Pageem et al。/em,1999)。 IpaD和IpaB定位的阴性对照分别是缺乏其不结合针尖的最后残基的IpaD蛋白(Espina等人,2006)和ipaD KO突变体其中IpaB不保留在尖端(Veenendaal等人,2007)。在这两种情况下,分泌的IpaD或IpaB不干扰测定
    4. 在4℃下搅拌孵育过夜。
    5. 加入750微升PBS的细菌,然后用1毫升PBS洗涤两次。所有进一步的步骤在黑暗中进行以保持二抗的荧光性质。微管或架子用铝箔覆盖。
    6. 重悬于250μlPBS + 4%BSA中,用1:500稀释的山羊CF647缀合的抗小鼠IgG抗体。
    7. 在4℃下搅拌1小时或更长时间(孵育时间可能持续过夜)。
    8. 向细菌中加入750μlPBS,然后用1ml PBS洗涤两次
    9. 将细菌沉淀重悬在500μlPBS中,并在FACS聚苯乙烯管中1:10稀释
    10. 例如,用四色FACS Calibur细胞计数器通过流式细胞术分析(在第三参考文献(Schiavolin等人,2013)的支持信息中可以找到结果的实例,可以在 > Mol.Microbiol。网站)。
    11. 使用的参数(无补偿):
      探测器
      电压
      模式
      FSC
      E02
      记录
      RSS
      366
      记录
      FL4
      800
      记录

食谱

  1. PBS + PFA 4%储备溶液(100ml)
    称重4g多聚甲醛在250ml锥形瓶中 加入80ml双蒸水
    加入50μl的1M NaOH
    添加磁条并关闭erlenmeyer
    在70℃加热直至完全溶解(溶液应变清澈)
    放在冰上,让溶液冷却至室温
    用双蒸水
    调节体积至90 ml 加入10 ml PBS 10x,并混合
    用0.2μm25mm尼龙注射器过滤器过滤溶液,并在15ml falcon管中等分 新鲜制备的PFA可以储存在-20℃用于进一步测定(在RT轻轻解冻)
  2. 刚果红琼脂平板
    用双蒸水制备30g/L TSB溶液,并溶解15g/L琼脂
    将培养基在120°C高压灭菌15分钟(如果您将培养基高压灭菌20分钟,糖会焦糖化,你的培养皿会变得更暗,甚至在37°C的细菌孵化后变得最暗)。
    添加刚果红,最终浓度为250μg/ml(当用保护性手套安全处理瓶子时,制备浓度为10mg/ml的双重蒸馏水中的储备溶液)。

致谢

该协议改编自Lee等人(2010)。本研究得到了比利时全国农业研究金(FRS-FNRS;公约3.4556.11)和欧洲共同体第七框架计划FP7/2011-2015(赠款协议第261742号)的资助。和A.M是来自比利时国家工业和农业(FRIA)的博士研究生的接受者。这项工作的一部分也得到Fonds Defay,Van Buuren和Héger-Masson基金会的支持。我们感谢A. Op de Beeck在执行FACS实验中的帮助。

参考文献

  1. Espina,M.,Olive,A.J.,Kenjale,R.,Moore,D.S.,Ausar,S.F.,Kaminski,R.W.,Oaks,E.V.,Middaugh,C.R.,Picking,W.D.and Picking,W.L。(2006)。 IpaD本地化到志贺氏菌的III型分泌系统针的尖端 。 74(8):4391-4400。
  2. Lee,P.C.,Stopford,C.M.,Svenson,A.G.and Rietsch,A。(2010)。 通过绿脓杆菌类型III控制效应物输出 分泌蛋白PcrG和PcrV。 Mol Microbiol 75(4):924-941。
  3. Page,A.L.,Ohayon,H.,Sansonetti,P.J.and Parsot,C。(1999)。 分泌的IpaB和​​IpaC侵袭素及其胞质伴侣IpgC是细胞间传播志贺氏菌所需的 。 1
  4. Schiavolin,L.,Meghraoui,A.,Cherradi,Y.,Biskri,L.,Botteaux,A.和Allaoui,A。(2013)。 对分泌控制中的志贺氏菌 III型针尖IpaD的功能洞察细胞接触。 Mol Microbiol 88(2):268-282
  5. Veenendaal,A.K.,Hodgkinson,J.L.,Schwarzer,L.,Stabat,D.,Zenk,S.F.and Blocker,A.J。(2007)。 III型分泌系统针尖复合物介导宿主细胞感测和translocon插入。 em> Mol Microbiol 63(6):1719-1730。
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How to cite this protocol: Schiavolin, L., Meghraoui, A. and Allaoui, A. (2013). Shigella IpaD and IpaB Surface Localizations. Bio-protocol 3(22): e975. DOI: 10.21769/BioProtoc.975; Full Text



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7/10/2014 11:17:54 AM  

Brooke Napier
Stanford University

The catalog # for the Congo Red from VWR is not correct.

Also, would be helpful to include the recipe for the CR agar plates to supplement this protocol.

7/11/2014 8:51:39 AM  

Lionel Schiavolin (Author)
Molecular Bacteriology Lab, Université Libre de Bruxelles, Belgium

Hi,

you'll find the CR from VWR at this URL:
https://uk.vwr.com/app/catalog/Product;jsessionid=6nvO4UhfRBxKo5Evg2WuAw**.node3?article_number=34140.184
I haven't found the same product from US VWR...

for CR agar plates' preparation :

- prepare a 30 g/l TSB solution with bidistilled water and dissolve 15 g/l agar.
- autoclave the medium for 15 min at 120°C (if you autoclave your medium for 20 min, sugar will caramelize and your plates will be darker, and even darkest after incubation with bacteria at 37°C)
- Add the CR at a final concentration of 250 µg/ml (stock solution is prepared in bidistilled water at a concentration of 10 mg/ml) when the bottle can be safely handled with a protective glove

I hope it'll help you.
Don't hesitate if you have more questions...

Lionel

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