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Shigella flexneri (S. flexneri) is a Gram-negative bacterium that causes gastroenteritis and shigellosis in humans. In order to establish and maintain an infection, S. flexneri utilises a type three secretion system (T3SS) to deliver virulence factors called effector proteins into the cytoplasm of host cells, facilitating e.g. uptake into the host cell and escape from the endosome. Secretion through the T3SS is tightly regulated and is usually triggered by host-cell contact, but can also be artificially stimulated in vitro. In this assay, the dye Congo red is used to induce T3SS-dependent secretion of S. flexneri (Parsot et al., 1995) and secreted proteins are concentrated from the culture supernatant by precipitation with trichloroacetic acid. The assay presented here can easily be adapted to the secretion analysis of other bacteria utilising a T3SS, such as Salmonella typhimurium, which constitutively secrete when grown at 37 °C (Collazo et al., 1995; Pegues et al., 1995), or pathogenic species of Yersinia, where secretion can be induced by calcium deprivation (Heesemann et al., 1986; Forsberg et al., 1987).

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Secretion Assay in Shigella flexneri
弗氏志贺菌的分泌试验

微生物学 > 微生物-宿主相互作用 > 细菌
作者: Jonathan Reinhardt
Jonathan ReinhardtAffiliation: Max-Planck-Institute for Infection Biology, Berlin, Germany
Bio-protocol author page: a1823
 and Michael Kolbe
Michael KolbeAffiliation: Max-Planck-Institute for Infection Biology, Berlin, Germany
For correspondence: kolbe@mpiib-berlin.mpg.de
Bio-protocol author page: a1824
Vol 4, Iss 22, 11/20/2014, 3251 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1302

[Abstract] Shigella flexneri (S. flexneri) is a Gram-negative bacterium that causes gastroenteritis and shigellosis in humans. In order to establish and maintain an infection, S. flexneri utilises a type three secretion system (T3SS) to deliver virulence factors called effector proteins into the cytoplasm of host cells, facilitating e.g. uptake into the host cell and escape from the endosome. Secretion through the T3SS is tightly regulated and is usually triggered by host-cell contact, but can also be artificially stimulated in vitro. In this assay, the dye Congo red is used to induce T3SS-dependent secretion of S. flexneri (Parsot et al., 1995) and secreted proteins are concentrated from the culture supernatant by precipitation with trichloroacetic acid. The assay presented here can easily be adapted to the secretion analysis of other bacteria utilising a T3SS, such as Salmonella typhimurium, which constitutively secrete when grown at 37 °C (Collazo et al., 1995; Pegues et al., 1995), or pathogenic species of Yersinia, where secretion can be induced by calcium deprivation (Heesemann et al., 1986; Forsberg et al., 1987).
Keywords: Shigella flexneri(福氏志贺菌), Type 3 Secretion System(3型分泌系统), Infection(感染), Bacteria(细菌), Microbe-Host Interaction(微生物-宿主相互作用)

[Abstract]

Materials and Reagents

  1. Tryptone soy agar (TSA) (Carl Roth, catalog number: CP70.1 )
  2. Shigella flexneri (strain M90T) on a Congo red tryptone soy agar plate
  3. 99% Trichloroacetic acid (TCA) (Carl Roth, catalog number: 8789 )
  4. Acetone (Merck KGaA, catalog number: 1000142511 )
  5. TRIZMA base (Sigma-Aldrich, catalog number: T1503 )
  6. Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L3771 )
  7. Bromophenol blue (Sigma-Aldrich, catalog number: B5525 )
  8. Glycerol (Sigma-Aldrich, catalog number: G7757 )
  9. 2-Mercaptoethanol (Sigma-Aldrich, catalog number: M3148 )
  10. LB medium (Carl Roth, catalog number: X968.2 ) (see Recipes)
  11. Tryptone soy agar plates (see Recipes)
  12. Sample buffer (see Recipes)
  13. Congo red stock solution (Sigma-Aldrich, catalog number: C6767 ) (see Recipes)
    Note: Used as 5 mg/ml stock solution.

Equipment

  1. 5 ml syringes (Henke-Sass, Wolf, catalog number: 5050.000V0 )
  2. 0.2 µm syringe filters (GE Healthcare, catalog number: 10462200 )
  3. Culture tubes
  4. 50 ml Erlenmeyer flasks
  5. 1.5 ml microtubes
  6. Microfuge
  7. 37 °C shaking incubator
  8. Laminar flow hood
  9. Spectrophotometer

Procedure

  1. Prepare a pre-culture by inoculating 2 ml of LB medium with a single colony of S. flexneri from a Congo red tryptone soy agar plate and grow overnight (~16 h) at 37 °C with agitation (180 rpm).
    Note: Ensure that the colonies used are secretion-competent, i.e. forming red colonies on the plate due to Congo red absorption (Payne and Finkelstein, 1977).
  2. Subculture by diluting 100 µl of the pre-culture in 10 ml of fresh LB medium (i.e. 1:100 dilution).
  3. Grow to OD600 (absorbance at 600 nm) of 0.3-0.4 at 37 °C with agitation.
    Note: In case genes from expression plasmids are to be induced, this should be done at OD600 of ~0.1.
  4. Add Congo red to a final concentration of 200 µg/ml (i.e. 400 µl of 5 mg/ml stock solution to 10 ml of culture).
  5. Grow for 2-3 h at 37 °C with agitation – the final OD600 of the culture should be between 2 and 3.
  6. Pellet cells by centrifugation for 10 min at 10,000 x g and 4 °C.
    Note: For the following TCA precipitation a supernatant volume corresponding to 1 ml culture of OD600=2 is used, meaning that centrifugation of 1.4 ml of culture in 1.5 ml tubes should be sufficient (considering the dead volume of the filter units in step 7).
  7. Transfer the supernatant to the 5 ml syringes and filter through 0.2 µm filters.
    Note: For maximum yield remove the piston from the syringe and add the filter. Transfer the supernatant into the syringe and replace the piston- the additional air will minimize the volume lost in the filter.
  8. Take a volume of filtered supernatant corresponding to 1 ml culture of OD600=2 and add ice-cold 99% trichloroacetic acid to a final concentration of 10% (v/v).
    Note: Congo red will turn blue at this point and precipitate as well.
  9. Transfer the tubes to -20 °C for 20 min.
  10. Centrifuge at top speed (12,000 x g to 16,000 x g) in a microfuge for 30 min at 4 °C.
  11. Carefully discard the supernatant and rinse the pellet with 1 ml ice-cold acetone.
  12. Centrifuge again (12,000 x g to 16,000 x g, 30 min, 4 °C).
  13. Carefully remove the supernatant and let the pellet air dry for 15 min.
  14. Resuspend the pellet in sample buffer.
    Note: The volume required may vary with the method of detection after electrophoresis: for analysis by Coomassie staining, resuspension in 25 µl to 100 µl will produce sufficiently strong bands.
  15. Analyse by SDS-PAGE through 12% acrylamide gels.

Representative data



Figure 1. Example of secretion from S. flexneri. TCA-precipitated pellets were resuspended in 25 µl of sample buffer and 20 µl loaded on a 12% polyacrylamide gel. CR = Congo red. ΔSpa33 is a T3SS-deficient mutant. IpaA, IpaB, IpaH, IpgD and IpaC are effector proteins. SepA is a T3SS-independent secreted protein and serves as an intrinsic loading control.

Note: IpaD may be discernible at 37 kDa, but is often obscured by Congo red. Congo red will return to its original color by soaking the gel in 100 mM Tris (pH 9.0) after Coomassie staining.

Recipes

  1. LB medium
    25 g LB medium powder
    Water to 1 L
    Note: It may be necessary to adjust the pH to 7.0-7.5 with a few drops of 1 M NaOH solution. This is usually not required, since LB medium is weakly buffered in this pH range.
    Autoclave the solution
  2. Tryptone soy agar plates
    20 g Tryptone soy agar powder
    0.05 g Congo red powder
    Water to 500 ml
    Note: It may be necessary to adjust the pH to 7.0-7.5 with a few drops of 1 M NaOH solution. This is usually not required, since TSA is weakly buffered in this pH range.
    Autoclave the solution
    Cool to ~50 °C and pour plates
  3. Sample buffer
    SDS-loading buffer with increased buffer capacity
    10% glycerol (v/v)
    360 mM Tris-HCl (pH 6.8)
    2% SDS (w/v)
    1.25% 2-mercaptoethanol (v/v)
    0.01% bromophenol blue (w/v)
  4. Congo red stock solution
    5 mg of Congo red powder per ml water
    Note that the dye content in the Congo red powder used here is ~40% and may differ between manufacturers. The stock solution is stable at room temperature for several months. When kept in the fridge or at -20 °C, Congo red may precipitate. Warm the solution to 37 °C to re-dissolve.

Acknowledgments

This work has been financially supported by the European Research Council under the European Community's Seventh Framework Programme (FP7/2007-2013) and a Ph.D. Scholarship from the International Max Planck Research School for Infectious Diseases and Immunology.
The protocol presented here has been adapted from Dohlich et al. (2014).

References

  1. Collazo, C. M., Zierler, M. K. and Galan, J. E. (1995). Functional analysis of the Salmonella typhimurium invasion genes invl and invJ and identification of a target of the protein secretion apparatus encoded in the inv locus. Mol Microbiol 15(1): 25-38.
  2. Dohlich, K., Zumsteg, A. B., Goosmann, C. and Kolbe, M. (2014). A substrate-fusion protein is trapped inside the Type III Secretion System channel in Shigella flexneri. PLoS Pathog 10(1): e1003881.
  3. Forsberg, A., Bolin, I., Norlander, L. and Wolf-Watz, H. (1987). Molecular cloning and expression of calcium-regulated, plasmid-coded proteins of Y. pseudotuberculosis. Microb Pathog 2(2): 123-137.
  4. Heesemann, J., Gross, U., Schmidt, N. and Laufs, R. (1986). Immunochemical analysis of plasmid-encoded proteins released by enteropathogenic Yersinia sp. grown in calcium-deficient media. Infect Immun 54(2): 561-567.
  5. Parsot, C., Menard, R., Gounon, P. and Sansonetti, P. J. (1995). Enhanced secretion through the Shigella flexneri Mxi-Spa translocon leads to assembly of extracellular proteins into macromolecular structures. Mol Microbiol 16(2): 291-300.
  6. Payne, S. M. and Finkelstein, R. A. (1977). Detection and differentiation of iron-responsive avirulent mutants on Congo red agar. Infect Immun 18(1): 94-98.
  7. Pegues, D. A., Hantman, M. J., Behlau, I. and Miller, S. I. (1995). PhoP/PhoQ transcriptional repression of Salmonella typhimurium invasion genes: evidence for a role in protein secretion. Mol Microbiol 17(1): 169-181.

材料和试剂

  1. 胰蛋白胨大豆琼脂(TSA)(Carl Roth,目录号:CP70.1)
  2. 在刚果红胰蛋白胨大豆琼脂平板上的 (菌株M90T)
  3. 99%三氯乙酸(TCA)(Carl Roth,目录号:8789)
  4. 丙酮(Merck KGaA,目录号:1000142511)
  5. TRIZMA碱(Sigma-Aldrich,目录号:T1503)
  6. 十二烷基硫酸钠(SDS)(Sigma-Aldrich,目录号:L3771)
  7. 溴酚蓝(Sigma-Aldrich,目录号:B5525)
  8. 甘油(Sigma-Aldrich,目录号:G7757)
  9. 2-巯基乙醇(Sigma-Aldrich,目录号:M3148)
  10. LB培养基(Carl Roth,目录号:X968.2)(参见Recipes)
  11. 胰蛋白胨大豆琼脂平板(见Recipes)
  12. 示例缓冲区(参见配方)
  13. 刚果红贮备液(Sigma-Aldrich,目录号:C6767)(参见Recipes)
    注意:用作5mg/ml储备溶液。

设备

  1. 5ml注射器(Henke-Sass,Wolf,目录号:5050.000V0)
  2. 0.2微米注射器过滤器(GE Healthcare,目录号:10462200)
  3. 文化管
  4. 50ml锥形瓶中
  5. 1.5 ml微管
  6. Microfuge
  7. 37℃振荡培养箱
  8. 层流罩
  9. 分光光度计

程序

  1. 准备预培养通过接种2ml LB培养基与单个集落的。 来自刚果红胰蛋白胨大豆琼脂平板,并在37℃下伴随搅拌(180rpm)生长过夜(〜16h)。
    注意:确保所使用的菌落具有分泌能力,即由于刚果红吸收在板上形成红色菌落(Payne和Finkelstein,1977)。
  2. 通过在10ml新鲜LB培养基(即1:100稀释)中稀释100μl预培养物进行传代培养。
  3. 在37℃下伴随搅拌生长至OD 600(在600nm处的吸光度)为0.3-0.4。
    注意:如果要诱导来自表达质粒的基因,应该在OD 600 em>
  4. 加入刚果红至最终浓度为200μg/ml(即,将400μl5mg/ml储备溶液加至10ml培养物中)。
  5. 在37℃下搅拌生长2-3小时,培养物的最终OD 600应在2和3之间。
  6. 通过在10,000×g和4℃下离心10分钟来沉淀细胞 注意:对于以下TCA沉淀,使用对应于1ml培养物的OD 600 = 600的上清液体积,这意味着离心的1.4ml培养物在1.5ml管中应该足够了(考虑到步骤7中过滤器单元的死体积)。
  7. 转移上清液到5ml注射器和通过0.2微米过滤器过滤。
    注意:为了最大产量,从注射器中取出活塞并添加过滤器。将上清液转移到注射器中并更换活塞 - 额外的空气将最小化过滤器中损失的体积。
  8. 取一定体积的对应于1ml OD 600 = 2的培养物的过滤的上清液,并加入冰冷的99%三氯乙酸至终浓度为10%(v/v)。
    注意:刚果红在这一点上会变成蓝色,也会沉淀。
  9. 将管转移至-20℃20分钟。
  10. 在微量离心机中在4℃下以最高速度(12,000×g至16,000×g)离心30分钟。
  11. 小心弃去上清液,并用1ml冰冷的丙酮冲洗沉淀
  12. 再次离心(12,000×g至16,000×g,30分钟,4℃)。
  13. 小心地除去上清液,让颗粒空气干燥15分钟
  14. 将沉淀重悬在样品缓冲液中。
    注意:电泳后检测方法可能会有所不同:对于考马斯染色分析,25μl至100μl的重悬将产生足够强的条带。
  15. 通过SDS-PAGE通过12%丙烯酰胺凝胶分析

代表数据



图1.来自 S的分泌示例。 flexneri 。将TCA沉淀的沉淀重悬于25μl样品缓冲液中,并将20μl上样于12%聚丙烯酰胺凝胶上。 CR =刚果红。 ΔSpa33是T3SS缺陷型突变体。 IpaA,IpaB,IpaH,IpgD和IpaC是效应蛋白。 SepA是一种不依赖T3SS的分泌蛋白,作为内在的加载控制
注意:IpaD在37kDa可能是可辨别的,但常常被刚果红模糊。 刚果红将通过在考马斯染色后将凝胶浸泡在100mM Tris(pH 9.0)中而恢复其原始颜色。

食谱

  1. LB培养基
    25g LB中等粉末
    水至1 L
    注意:可能需要用几滴1M NaOH溶液将pH调节至7.0-7.5。 这通常不是必需的,因为LB培养基在该pH范围内弱缓冲。
    高压灭菌溶液
  2. 胰蛋白胨大豆琼脂平板
    20g胰蛋白胨大豆琼脂粉末
    0.05g刚果红粉末
    水至500 ml
    注意:可能需要用几滴1M NaOH溶液将pH调节至7.0-7.5。 这通常不是必需的,因为TSA在该pH范围内弱缓冲。
    高压灭菌解决方案
    冷却至〜50°C,倒入板
  3. 示例缓冲区
    具有增加的缓冲容量的SDS加载缓冲液
    10%甘油(v/v) 360 mM Tris-HCl(pH 6.8)
    2%SDS(w/v)
    1.25%2-巯基乙醇(v/v)
    0.01%溴酚蓝(w/v)
  4. 刚果红贮液
    5毫克刚果红粉/毫升水
    注意,这里使用的刚果红粉末中的染料含量为〜40%,并且可能在制造商之间不同。 储备溶液在室温下稳定几个月。 当保存在冰箱或-20°C时,刚果红可能沉淀。 将溶液温热至37℃以重新溶解。

致谢

这项工作得到了欧洲研究理事会在欧洲共同体第七框架计划(FP7/2007-2013)的资助下的资助,并获得博士学位。 来自国际传染病和免疫学硕士研究学院的奖学金。
这里提出的协议已经改编自Dohlich等人(2014)。

参考文献

  1. Collazo,C.M.,Zierler,M.K.and Galan,J.E。(1995)。 鼠伤寒沙门氏菌入侵基因的功能分析 invl < em>和< em> invJ</em>和鉴定编码在inv inv基因座中的蛋白质分泌装置的靶标。 ):25-38。
  2. Dohlich,K.,Zumsteg,A.B.,Goosmann,C.and Kolbe,M.(2014)。 底物融合蛋白被捕获在 Shigella flexneri 中的III型分泌系统通道内。 PLoS Pathog 10(1):e1003881。
  3. Forsberg,A.,Bolin,I.,Norlander,L.and Wolf-Watz,H。(1987)。 Y的钙调节,质粒编码蛋白的分子克隆和表达。假结核。 Microb Pathog 2(2):123-137。
  4. Heesemann,J.,Gross,U.,Schmidt,N。和Laufs,R。(1986)。 由肠致病性耶尔森氏菌发布的质粒编码蛋白的免疫化学分析。在钙缺乏的培养基中生长。 Infect Immun 54(2):561-567。
  5. Parsot,C.,Menard,R.,Gounon,P。和Sansonetti,P.J。(1995)。 通过 Shigella flexneri Mxi-Spa translocon增强分泌导致组装 胞外蛋白质转化为大分子结构。 Mol Microbiol 16(2):291-300。
  6. Payne,S.M。和Finkelstein,R.A。(1977)。 刚果红琼脂上铁应答性无毒突变体的检测和分化。 Infect Immun 18(1):94-98。
  7. Pegues,D.A.,Hantman,M.J.,Behlau,I。和Miller,S.I。(1995)。 PhoP/PhoQ转录抑制鼠伤寒沙门氏菌入侵基因: 在蛋白质分泌中的作用。 Mol Microbiol 17(1):169-181。
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How to cite this protocol: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Reinhardt, J. and Kolbe, M. (2014). Secretion Assay in Shigella flexneri. Bio-protocol 4(22): e1302. DOI: 10.21769/BioProtoc.1302; Full Text
  2. Dohlich, K., Zumsteg, A. B., Goosmann, C. and Kolbe, M. (2014). A substrate-fusion protein is trapped inside the Type III Secretion System channel in Shigella flexneri. PLoS Pathog 10(1): e1003881.




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