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Design of a Transcription-based Secretion Activity Reporter (TSAR) for the Type III Secretion Apparatus of Shigella flexneri and Uses Thereof
   

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Cell Host & Microbe
Feb 2014

Abstract

Many gram-negative bacterial pathogens, including Shigella flexneri, are able to translocate bacterial proteins, dubbed effectors, across the host cell plasma membrane into the host cell cytosol using a syringe-like structure, the type three secretion apparatus (T3SA). While some bacteria use their T3SA to modulate their phagosomal environment (Salmonella spp.), establish pedestal structure to form microcolonies on the plasma membrane (Enteropathogenic Escherichi coli) or lyse their entry vacuole (Shigella spp.), they all have in common a tightly regulated activity of their T3SA. However, the tracking of the activity of the T3SA in infected cells and tissue has been difficult to perform. Using the property of MxiE-dependent promoters that are upregulated when the T3SA is active, we have recently designed a transcription-based secretion activity reporter (TSAR) that allows the following of the activity of the S. flexneri T3SA in real-time in tissue culture cells and in vivo using fast maturing GFP intrinsic fluorescence. Herein we describe the design of the TSAR and its application to fixed and live samples for microscopy and flow cytometry in a colonic epithelial cell model using TC7 tissue culture cells.

Keywords: Shigella (志贺菌), Transcriptional reporter (转录记者), Green Fluorescent Protein (绿色荧光蛋白), Type Three Secretion Apparatus (三型分泌器), Bacteril (菌)

Material and Reagents

  1. Transcription-based secretion activity reporter plasmids (pTSAR1.3, pTSAR1Ud2.1 or pTSAR1Ud2.4s)
    Note: Directly available under material transfer agreement (MTA) from Philippe Sansonetti’s laboratory.
  2. Petri dish of Tryptone Casein Soya (TCS) agar (BD Biosciences, catalog number: 236950 ) supplemented with 0.01% Congo red (CR) (SERVA Electrophoresis GmbH, catalog number: 27215.01 ) and the appropriate antibiotic
  3. TCS Broth (BD Biosciences, catalog number: 211825 )
  4. Ampicillin (MP Biomedicals, catalog number: 0 219452605 )
  5. Polylysine (Sigma-Aldrich, catalog number: P1274 )
  6. Human tissue culture cells such as as colonic epithelial TC7 cells (a clone of Caco-2 cells)
    Note: Only polarized epithelial cells permit efficient cell-to-cell spread of Shigella spp. We also recommend using Human cells because it is the sole natural host of Shigella, although most cell lines of other origins tested are also readily infected and could be used for practical reasons.
  7. DMEM (Life Technologies, catalog number: 31885 )
  8. FCS (Biowest, catalog number: S1810-100 )
  9. Penicillin/Streptomycin (Life Technologies, catalog number: 15140 )
  10. Non-essential amino acids (Life Technologies, catalog number: 11140 )
  11. 0.25% trypsin-EDTA (Life Technologies, catalog number: 25200-056 )
  12. Fibronectin from human plasma (Sigma-Aldrich, catalog number: F0895 ) (optional)
  13. HEPES (Life Technologies, catalog number: 15630-056 )
  14. Gentamicin (Sigma-Aldrich, catalog number: G1397 )
  15. Cell mask deep red (Life Technologies, catalog number: C10046 )
  16. Cytochalasin D (Sigma-Aldrich, catalog number: C2873 )
  17. Live imaging medium containing DMEMgfp-2 (Evrogen, catalog number: MC102)
  18. PFA (Electron Microscopy Sciences, catalog number: 15714 )
  19. Glycine (Sigma-Aldrich, catalog number: G7126 )
  20. Triton X-100 (Sigma-Aldrich, catalog number: T8787 ) (optional)
  21. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A 9647) (optional)
  22. Gelatin (Sigma-Aldrich, catalog number: G1393 ) (optional)
  23. Sodium azide (Sigma-Aldrich, catalog number: 0 8591 ) (optional)
  24. DAPI stock solution (Sigma-Aldrich, catalog number: D9542 ) (optional)
  25. MOWIOL (Sigma-Aldrich, catalog number: 81381 )
  26. 1,4-diazobicyclo-[2,2,2]-octaned (DABCO) (Sigma-Aldrich, catalog number: D27802 )
  27. Glycerol (VWR International, catalog number: 24388.295 ) alternatively Prolong mounting medium (Life Technologies, catalog number: P36930 )
  28. 10x phosphate-buffer saline (PBS) (see Recipes)
  29. PBS/10 µg/ml polylysine (see Recipes)
  30. DMEM-HEPES (see Recipes)
  31. Chase medium (see Recipes)
  32. Live imaging medium (see Recipes)
  33. PBS/4% PFA (see Recipes)
  34. PBS/100 mM glycine (see Recipes)
  35. 5,000x DAPI stock solution (see Recipes)
  36. Mounting medium (see Recipes)
  37. Growth medium for TC7 cells (see Recipes)

Equipment

  1. 35 mm petri dish with glass bottom or 8-wells microplate (Ibidi®, catalog numbers: 81158 and 80826 , respectively) for live microscopy
  2. 24-well plate
  3. Coverslips no. 1.5, 12 mm in diameter (Harvard Apparatus, catalog number: 64-0712 )
  4. Laminar flow hood for cell culture
  5. Heating water bath
  6. Tabletop centrifuge for 1.5-2 ml Eppendorf tubes
  7. Tabletop centrifuge with plate-holding rotor
  8. CO2 incubator for cell culture
  9. Tweezers

Procedure

  1. Introductory comments concerning the design of the TSAR
    1. The design of the TSAR vector was described in detail elsewhere (Campbell-Valois et al., 2014). In brief, expression of a fast maturing variant of GFP (e.g. GFPmut2, GFPmut3 or GFPsfm2) was placed under the control of the promoter of ipaH7.8, a virulence gene regulated by the activity of the Type Three Secretion Apparatus (T3SA). Thus, upon activation of the T3SA, GFP becomes transcribed and translated and allows the monitoring of the T3S system activity (Figure 1).
    2. Critical elements to optimize the expression level and the signal of the reporter were found to be translation initiation (Shine and Dalgarno sequence and the codon bias of the reporter gene, particularly in the region immediately downstream of the start codon) and the intrinsic properties of the reporter protein used (e.g. brightness, maturation rate etc.). The half-life of  GFP, which is normally several hours, could be reduced to around 40 min by fusing the ssrA peptide to the carboxy-terminus, which rendered GFP susceptible to ClpX protease-mediated degradation. Using this approach, we improved the capacity of the TSAR to measure the interruption of the T3SA activity in real-time. Another essential element of the TSAR plasmids is the constitutive expression, using the rpsM promoter, of a second fluorescent protein whose excitation and emission spectra are compatible with the GFP expressed from the T3SA-dependent promoter. The combination of both promoters allowed for following the bacteria in various conditions and assays (microscopy multiple fluorescence, flow cytometer etc.) (Figure 1).
    3. Using the above mentioned guidelines to optimize the maturation rate, translation initiation, codon usage and half-life as well as selecting the best reporter protein for the desired assay (e.g. RFP, lux operon luciferase, etc.), other reporter systems derived from the original GFP-based TSAR could easily be designed. Here we provide three protocols using the GFP-based TSAR with S. flexneri to assess T3SA activity for fixed samples, live microscopy and flow cytometry.


      Figure 1. Plasmid maps.
      Map of pTSAR1Ud2.1 designed to perform double immunofluorescence followed by microscopic observations A. Map of pTSAR1Ud.4s used in microscopy and particularly live microscopy B. Map of pTSAR1.3 used for FACS applications C.

  2. Fixed samples microscopy
    1. 36-48 h before the experiment, detach a confluent TC7 cells monolayer with 0.25% trypsin/EDTA and distribute TC7 cells on coverslips no. 1.5 placed in wells of a 24-well plate in their normal growth medium. To follow entry events, seed 5 x 104 cells/well (approximately 50-75% confluence the day of the experiment). To favor cell-to-cell spread during longer infection times, seed 1 x 105 cells/well. (Optional) Fibronectin-coated coverslips can be used to permit faster attachment of the cells, but in this case, the number of cells seeded must be reduced about 25-50% to obtain confluence similar to what is described for non-coated coverslips.
    2. The day before the experiment, a CR–positive colony harboring pTSAR1ud2.1 or pTSAR1Ud2.4s is picked from a CR-containing TCS ampicillin plate and used to inoculate one tube of 8 ml TCS broth supplemented with Amp and incubated overnight at 30 °C with shaking.
      Notes:
      1. CR induces T3SA activation and effector secretion; colonies with an active T3SA therefore display a red center where CR has accumulated while bacteria that have lost the virulence plasmid show up as larger white colonies (Figure 2).
      2. Strains harboring pTSAR1Ud2.1 are used in place of pTSAR1Ud2.4 for multiple labeling experiments because it allows the use of the red laser/Cy3 filter for immunofluorescence.


      Figure 2. Congo red positive (CR+) versus congo red negative (CR-) colonies.
        Wt and plasmid cured M90T derived strains were streaked side-by-side on an agar-containing TCS and congo red plate to show their differing phenotypes. Wt colonies on the left are forming small and CR+ colonies (displaying a red center). Plasmid cured (BS176 strain) are forming large CR- colonies (white).

    3. The next morning, the ON culture is subcultured 1:100 in a tube of 8.0 ml fresh TCS broth without antibiotics and incubated at 37 °C until the OD600 is approximately 1.0 (late exponential phase).
    4. 2 ml of bacterial culture (sufficient for the infection of 4 wells of a 24-wells plate) is centrifuged at 7,500 x g for 1 min and washed in 1 ml PBS. Repeat two other times the centrifugation and washing steps.
    5. Resuspend the bacterial pellet in 2 ml DMEM-HEPES at room temperature (RT).
    6. TC7 cells are washed 2 times with DMEM-HEPES (RT), after the washes place DMEM-HEPES at 37 °C for step B9.
    7. 500 µl of the DMEM-HEPES bacterial suspension is added to each well of the 24-well plate seeded with TC7 cells.
    8. Bacteria are brought into contact with the cells by centrifugation for 5 min at 450 x g at RT. Cells are maintained in contact with the bacterial suspension for approximately 10 min at RT in total. (Optional) if desirable, polylysine-coated bacteria can be used instead of centrifugation. Polylysine-coated bacteria can be prepared by incubating bacteria, which were washed once with 1 ml PBS (see step 4), in PBS/polylysine (10 µg/ml) for 10 min with gentle agitation and washing of the bacteria three times prior to infection. Entry efficiency is greatly enhanced using polylysine treatment, so a bacterial suspension with OD600 equal to 0.02-0.2 should be incubated with the cells at RT for 10 min without centrifugation.
    9. The bacteria suspension is aspirated and replaced with 500 µl DMEM-HEPES preheated to 37 °C.
    10. Incubate between 15 to 30 min in a 37 °C cell culture incubator with 5% CO2.
    11. Aspirate medium and add the chase medium for the desired time. To study cell-to-cell spread in TC7 cells, a chase of 210 min is ideal.  
    12. At the desired time, wash the cell monolayer once with PBS.
    13. Fix for 10 min with diluted 200 µl PBS/4% PFA at RT.
    14. Aspirate the fixative and incubate 5 min in 200 µl PBS/glycine to quench the PFA.
    15. (Optional) DAPI staining (0.2 µg/ml) can be performed in PBS by incubating the cells during 20 min.
    16. Wash 3 times with PBS.
    17. (Optional) If desired, cells can be permeabilized to perform immunofluorescence labeling following assay-specific protocols. We routinely use 0.1% Triton X-100 for 10 min to permeabilize cells while maintaining GFP intrinsic fluorescence. Saponin is also usable for this application, while methanol/acetone-based methods are not. Blocking is performed in PBS/1%BSA/0.2% gelatin. Standard immunofluoresence procedure should then be used to stain the coverslip.
      Note that in this case, the DAPI staining is performed, as described above, during incubation with the secondary antibodies.
    18. Take out the coverslips from the plate with a fine pair of tweezers, briefly immerse in distilled water and remove excess water using absorbent paper.
    19. Mount on a slide using 10 µl of pre-warmed (37 °C) mounting medium.
    20. Let dry without disturbing the CS for at least one hour at room temperature.
    21. Store at 4 °C until ready to observe.
    22. The GFP signal in the bacteria is visualized using a GFP or FITC filter or with the 488 nm band of an argon laser. The Cerulean signal is visualized using a CFP filter or with the 458 nm band of an argon laser. mCherry is visualized using a Cy3 filter or with a 568 nm laser. We do not recommend using DsRed in conjunction with GFP in microscopy applications.

  3. Live microscopy
    1. TC7 cells are passed at the desired density in regular growth medium 48 h before the experiment in a 35 mm dish with a glass bottom or in an 8-well microplate, if several conditions are to be tested in parallel.
      Note: We have also used HeLa and LLC-MK2 cells.
      A bacterial suspension of bacteria harboring the pTSAR1Ud2.4s is prepared as above and polylysine coated bacteria are routinely used (see step B8), particularly to monitor entry as entry efficiency is greatly enhanced by this pretreatment. Centrifugation-induced infection without precoating bacteria with polylysine can also be performed by carefully securing the dish or microplate to the plate holder of the centrifuge (see steps B3-8).
      Note: Entry of polylysin coated bacteria can be blocked by pretreating cells with 2 µM cytochalasin D, while adhesion to host cells is maintained. Entry can then be triggered within approximately 20 min on HeLa cells by aspirating the cytochalasin D containing media, washing the cell monolayer once and replacing the medium with live imaging medium. This last step can be performed on the microscope stage to allow monitoring of early events leading to entry.
    2. Perform entry and chase of bacterial infection, as described (see steps B9-11), by adjusting incubation time as desired.
    3. (Optional) The plasma membrane of infected cells can be labeled immediately prior to microscopic observations using Cell Mask Deep Red. The staining is performed by incubating the cells for 5 min with DMEM-HEPES containing 1:1,000 dilution of the Cell Mask. Stained cells are washed three times with DMEM-HEPES and proceed to step C4 just prior to imaging.
    4. Wash the cells monolayer once with 2 ml live imaging medium or PBS, aspirate, and add 2 ml live-imaging medium to the cell monolayer.
    5. Transfer the infected cells to the stage of the microscope, which ideally should be equipped with a controlled atmosphere chamber (e.g. 37 °C and 5% CO2).
    6. To track bacteria, acquire at least 0.5-1 image per minute in fast acquisition mode (using 2 x 2 binning for pixels, for example, can reduce acquisition time). GFP, mCherry and Cell Mask Deep Red can be imaged simultaneously on most microscopic systems used using Cy3 and Cy5 filters, respectively.

  4. Preparation of samples for flow cytometry (FC) analysis
    1. Pass TC7 cells as above but use one to two wells of a 6-well plate per condition or kinetic point that is to be studied. Distribute 1 x 105-2 x 105 cells per well according to the level of confluence desired for the experiment, taking into account that higher confluence will favor cell-to-cell spread but reduce the initial entry of bacteria.
    2. Grow bacteria as described above. Strains harboring pTSAR 1.3 are ideal for flow cytometry (FC) because constitutive expression of the DsRed MS165 from the ribosomal rpsM-promoter allows tracking non-secreting bacteria.
      Note: In this protocol, we suggest using polylysine-coated S. flexneri to favor entry and maximize recovery of bacteria for FC analyses (see step B8).
    3. TC7 cells are washed two times with DMEM-HEPES (RT).
    4. 2 ml of the DMEM-HEPES bacterial suspension is added to each well of the 24-wells plate containing TC7 cells.
    5. Induction of infection is performed as described above (see steps B3-8).
    6. The infection is allowed to proceed for the desired time (see steps B9-11).
    7. Bacteria are recovered from the infected cells by detergent lysis, fixation and centrifugation as described in (Aussel et al., 2011). Briefly, cells are washed once in PBS, lysed in 220 µl PBS/0.1% Triton X-100, scraped off the plate, and fixed during 60 min by the addition of 440 µl PBS/3% PFA. Bacteria are then transferred into a 1.5 ml tube and recovered by two centrifugation steps: 5 min at 200 x g to pellet the cellular nuclei; recover the supernatant without disturbing the pellet and centrifuge the supernatant for 10 min at 16,000 x g.
    8. The pellet is recovered with 50 µl PBS/100 mM glycine.
    9. After 10 min at RT, samples are further diluted 1:10 in PBS.
    10. (Optional) samples at this stage can be kept at 4 °C, protected from light, for at least three weeks.
    11. Samples are transferred to FC tubes and can be further diluted so that approximately 1,000-3,000 events are recorded per second.
    12. To adjust the compensation and other FC parameters, it is practical to use dilutions of bacterial cultures used for infection and non-labeled and singly labeled strains that were previously fixed with PFA as indicated above.

Notes

  1. Results are usually highly reproducible. However, to ensure that the ratio of secreting bacteria to not-secreting bacteria is constant at the given time post-challenge under scrutiny, it is recommended to ensure that the infected cells be studied at a similar level of confluence because when bacteria are unable to perform cell-to-cell spread efficiently, the proportion of immobile, and hence not-secreting bacteria, increases. Similarly, bacteria subculturing prior to infection must be performed in highly controlled conditions, for example, by taking care of stopping incubation at similar OD600 each time an experiment is repeated.

Recipes

  1. 10x phosphate-buffer saline (PBS)
    Mix 80 g of NaCl with 2 g of KCl, 14.4 g of Na2HPO4, 2.4 g of KH2PO4
    Adjust pH to 7.4 with NaOH
    Add distilled H2O to 1,000 ml
    Filter or autoclave
    Stored at RT
    For all applications, a 1x solution is used
  2. PBS/10 µg/ml polylysine
    PBS
    1:1,000 of the polylysine stock solution (10 mg/ml in water, filter sterilized and stored at -20 °C)
  3. DMEM-HEPES
    DMEM
    20 mM HEPES
  4. Chase medium
    DMEM
    5% FBS
    50 µg/ml gentamicin
  5. Live imaging medium
    DMEMgfp-2
    5% FBS
    50 µg/ml gentamicin
    Note: Ringer’s buffer can be used in place of the live imaging medium, particularly for infection of short duration.
  6. PBS/4% PFA
    10 ml PFA (32%)
    8 ml 10x PBS
    Add distilled H2O to 80 ml
    Aliquot and stored at -20 °C
    Discard aliquot after a week at 4 °C
  7. PBS/100 mM Glycine
    0.375 g glycine
    5 ml 10x PBS
    Add H2O to 50 ml
    Filter sterilized (0.2 µm)
    Stored at 4 °C
  8. 5,000x DAPI stock solution
    1 ml H2O
    1 mg DAPI
  9. Mounting medium
    Note: Recipe from the University of Rochester Medical Center, Michael Mastrangelo, Eric Yehling; for details see http://www.urmc.rochester.edu/confocal-conventional-microscopy/documents/Mowiol52810.pdf.
    24 g analytical grade glycerol
    9.6 g Mowiol 4-88
    24 ml distilled H2O
    48 ml 0.2 M Tris buffer (pH 8.5)
    To dissolve completely assemble the solution in a 500 ml flask and stir on a heating plate at 60-70 °C for 4-5 h
    Aliquot in 20-40 ml per 50 ml conical
    Centrifuge at 5,000 x g for 15 min to remove undissolved residues and carefully recover the supernatant
    Add 2.5% 1,4-diazobicyclo-[2,2,2]-octane (DABCO) as anti bleaching agent
    Aliquot in 15 or 50 ml conicals
    Stored at -20 °C for long-term storage
  10. Growth medium for TC7 cells
    DMEM
    20% FCS
    1x Penicillin/Streptomycin
    1x Non-essential amino acids

Acknowledgments

We are grateful to Claude Parsot for his comments on the manuscript. FXCV was a CIHR, EMBO, Marie-Curie-IRG and FRM fellow. PS was an EMBO and Marie Curie-IEF fellow while PJS is a HHMI senior scholar. This work was supported by the ERC (PJS Advanced Grant, no. 232798).

References

  1. Aussel, L., Zhao, W., Hebrard, M., Guilhon, A. A., Viala, J. P., Henri, S., Chasson, L., Gorvel, J. P., Barras, F. and Meresse, S. (2011). Salmonella detoxifying enzymes are sufficient to cope with the host oxidative burst. Mol Microbiol 80(3): 628-640.
  2. Campbell-Valois, F. X., Schnupf, P., Nigro, G., Sachse, M., Sansonetti, P. J. and Parsot, C. (2014). A fluorescent reporter reveals on/off regulation of the Shigella type III secretion apparatus during entry and cell-to-cell spread. Cell Host Microbe 15(2): 177-189.

简介

包括灵芝氏菌在内的许多革兰氏阴性细菌病原体能够使用注射器样结构将类型三分泌物穿过宿主细胞质膜转运细菌蛋白质(配体效应子)到宿主细胞胞质溶胶中装置(T3SA)。虽然一些细菌使用它们的T3SA调节它们的吞噬体环境(沙门氏菌

spp 。),建立基质结构以在质膜上形成微集落(Enteropathogenic < em>)或溶解它们的进入泡(志贺氏菌属。),它们都具有共同的严格调节的他们的T3SA的活性。然而,T3SA在感染的细胞和组织中的活性的跟踪一直难以进行。使用在T3SA是活性时上调的MxiE依赖性启动子的性质,我们最近设计了基于转录的分泌活性报道分子(TSAR),其允许以下的S的活性。使用快速成熟的GFP内在荧光,在组织培养细胞中实时地和体内实时分析灵敏度。在这里我们描述TSAR的设计及其应用于固定和活体样品的显微镜和流式细胞仪在结肠上皮细胞模型使用TC7组织培养细胞。

关键字:志贺菌, 转录记者, 绿色荧光蛋白, 三型分泌器, 菌

材料和试剂

  1. 基于转录的分泌活性报告质粒(pTSAR1.3,pTSAR1Ud2.1或pTSAR1Ud2.4s)
    注意:根据材料转让协议(MTA)直接提供,来自Philippe Sansonetti的实验室。
  2. 补充有0.01%刚果红(CR)(SERVA Electrophoresis GmbH,目录号:27215.01)和合适的抗生素的胰蛋白胨酪蛋白大豆(TCS)琼脂(BD Biosciences,目录号:236950)
  3. TCS肉汤(BD Biosciences,目录号:211825)
  4. 氨苄青霉素(MP Biomedicals,目录号:0219452605)
  5. 聚赖氨酸(Sigma-Aldrich,目录号:P1274)
  6. 人组织培养细胞如结肠上皮TC7细胞(Caco-2细胞的克隆)
    注意:只有极化的上皮细胞允许志贺氏菌属的有效的细胞到细胞的传播。 我们还建议使用人类细胞,因为它是志贺氏菌的唯一自然宿主,虽然其他测试来源的大多数细胞系也容易感染,可能由于实际原因使用。
  7. DMEM(Life Technologies,目录号:31885)
  8. FCS(Biowest,目录号:S1810-100)
  9. 青霉素/链霉素(Life Technologies,目录号:15140)
  10. 非必需氨基酸(Life Technologies,目录号:11140)
  11. 0.25%胰蛋白酶-EDTA(Life Technologies,目录号:25200-056)
  12. 来自人血浆的纤连蛋白(Sigma-Aldrich,目录号:F0895)(任选)
  13. HEPES(Life Technologies,目录号:15630-056)
  14. 庆大霉素(Sigma-Aldrich,目录号:G1397)
  15. 细胞膜深红色(Life Technologies,目录号:C10046)
  16. 细胞松弛素D(Sigma-Aldrich,目录号:C2873)
  17. 含有DMEM gfp -2(Evrogen,目录号:MC102)的活成像培养基
  18. PFA(Electron Microscopy Sciences,目录号:15714)
  19. 甘氨酸(Sigma-Aldrich,目录号:G7126)
  20. Triton X-100(Sigma-Aldrich,目录号:T8787)(可选)
  21. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A 9647)(任选)
  22. 明胶(Sigma-Aldrich,目录号:G1393)(可选)
  23. 叠氮化钠(Sigma-Aldrich,目录号:08591)(可选)
  24. DAPI储备溶液(Sigma-Aldrich,目录号:D9542)(可选)
  25. MOWIOL(Sigma-Aldrich,目录号:81381)
  26. 1,4-二氮杂双环 - [2,2,2] - 辛烷(DABCO)(Sigma-Aldrich,目录号:D27802)
  27. 甘油(VWR International,目录号:24388.295)或长寿命封固剂(Life Technologies,目录号:P36930)
  28. 10x磷酸盐缓冲盐水(PBS)(参见配方)
  29. PBS /10μg/ml聚赖氨酸(见Recipes)
  30. DMEM-HEPES(参见配方)
  31. Chase介质(见配方)
  32. 活成像介质(见配方)
  33. PBS/4%PFA(参见配方)
  34. PBS/100mM甘氨酸(参见配方)
  35. 5,000x DAPI储备溶液(见配方)
  36. 安装介质(参见配方)
  37. TC7细胞的生长培养基(参见配方)

设备

  1. 带有玻璃底或35孔培养皿(分别为目录号:81158和80826)的8孔微量平板(分别为Ibidi ,目录号:81158和80826)
  2. 24孔板
  3. Coverslips no。 1.5,直径12mm(Harvard Apparatus,目录号:64-0712)
  4. 用于细胞培养的层流罩
  5. 加热水浴
  6. 台式离心机用于1.5-2 ml Eppendorf管
  7. 带托板转子的台式离心机
  8. CO 2细胞培养箱中培养
  9. 镊子

程序

  1. 关于TSAR设计的介绍性意见
    1. TSAR载体的设计在别处详细描述(Campbell-Valois等人,2014)。简言之,将GFP的快速成熟变体(例如GFPmut2,GFPmut3或GFPsfm2)的表达置于ipaH7.8启动子的控制下,所述启动子是毒力基因调节的通过三型分泌装置(T3SA)的活性。因此,在激活T3SA后,GFP被转录和翻译,并允许监测T3S系统活性(图1)。
    2. 发现优化表达水平和报道分子信号的关键元件是翻译起始(Shine和Dalgarno序列和报告基因的密码子偏好,特别是在紧接起始密码子下游的区域中)和内在特性使用的报道蛋白(例如,亮度,成熟率等)。 半衰期通常将几个小时的GFP通过将ssrA肽融合到羧基末端可以减少到约40分钟,这使得GFP易受ClpX蛋白酶介导的降解的影响。使用这种方法,我们提高了TSAR的实时测量T3SA活动中断的能力。 TSAR质粒的另一个基本要素是使用rpsM启动子的第二荧光蛋白的组成型表达,其激发和发射光谱与从T3SA依赖性启动子表达的GFP相容。两种启动子的组合允许在各种条件和测定(显微镜多荧光,流式细胞仪等)中遵循细菌(图1)。(图1)。
    3. 使用上述指南优化成熟率,翻译起始,密码子使用和半衰期,以及为所需测定选择最佳报告蛋白(例如 RFP,lux 操纵子荧光素酶,等),可以容易地设计源自原始基于GFP的TSAR的其他报告基因系统。在这里,我们提供使用基于GFP的TSAR与 S的三个协议。 flexneri 来评估固定样品,活体显微镜和流式细胞术的T3SA活性

      图1.质粒图谱。设计用于进行双重免疫荧光,然后进行显微观察的pTSAR1Ud2.1的图谱A.显微镜,特别是实时显微镜中使用的pTSAR1Ud.4s的图谱B.用于FACS应用的pTSAR1.3的图谱C 。

  2. 固定样品显微镜
    1. 在实验前36-48小时,用0.25%胰蛋白酶/EDTA分离融合的TC7细胞单层,并将TC7细胞分布在盖玻片上。 1.5置于其正常生长培养基中的24孔板的孔中。为了跟踪进入事件,种子为5×10 4个细胞/孔(实验当天大约50-75%汇合)。为了在较长的感染时间期间有利于细胞间扩散,以1×10 5个细胞/孔种子。 (任选的)纤连蛋白包被的盖玻片可用于允许更快的细胞附着,但是在这种情况下,接种的细胞数目必须减少约25-50%,以获得类似于对于未包被的盖玻片描述的汇合。
    2. 在实验前一天,从含有CR的TCS氨苄青霉素板中挑取含有pTSAR1ud2.1或pTSAR1Ud2.4s的CR阳性菌落,并用于接种一管补充有Amp的8ml TCS肉汤,并在30℃温育过夜与摇动。
      注意:
      1. CR诱导T3SA激活和效应物分泌;具有活性T3SA的菌落因此显示红色中心,其中CR已经积累,而已经失去毒力质粒的细菌显示为更大的白色菌落(图2)。
      2. 具有pTSAR1Ud2.1的菌株代替pTSAR1Ud2.4用于多重标记实验,因为它允许使用红色激光/Cy3滤光片进行免疫荧光。


      图2.刚果红阳性(CR +)对刚果红色阴性(CR-)菌落。将Wt和质粒固化的M90T衍生菌株在含琼脂的TCS和刚果红板上平行划线以显示它们不同的表型。左侧的Wt集落形成小和CR +集落(显示红色中心)。质粒固化(BS176菌株)形成大的CR-菌落(白色)
    3. 第二天早上,将ON培养物在不含抗生素的8.0ml新鲜TCS肉汤的管中以1:100传代培养,并在37℃温育直至OD 600大约为1.0(指数晚期) br />
    4. 将2ml细菌培养物(足以感染24孔板的4个孔)以7,500×g离心1分钟,并在1ml PBS中洗涤。 重复另外两次离心和洗涤步骤。
    5. 在室温(RT)下将细菌沉淀重悬在2ml DMEM-HEPES中
    6. TC7细胞用DMEM-HEPES(RT)洗涤2次,洗涤后,在37℃下将DMEM-HEPES置于步骤B9。
    7. 将500μlDMEM-HEPES细菌悬浮液加入接种有TC7细胞的24孔板的每个孔中。
    8. 通过在RT下以450x g离心5分钟使细菌与细胞接触。使细胞与细菌悬浮液在总体上保持接触约10分钟。 (任选的)如果需要,可以使用聚赖氨酸包被的细菌代替离心。聚赖氨酸包被的细菌可以通过温育细菌孵育细菌来制备,所述细菌用PBS /聚赖氨酸(10μg/ml)中的1ml PBS(参见步骤4)洗涤一次,温和搅拌10分钟,洗涤细菌3次,然后感染。使用聚赖氨酸处理大大增强了进入效率,因此OD 600等于0.02-0.2的细菌悬浮液应与细胞在室温下孵育10分钟,而不进行离心。
    9. 吸出细菌悬浮液并用预热至37℃的500μlDMEM-HEPES替换
    10. 在具有5%CO 2的37℃细胞培养箱中孵育15至30分钟。
    11. 吸出培养基并添加追踪培养基所需的时间。为了研究TC7细胞中的细胞到细胞的扩散,追踪210分钟是理想的。  
    12. 在所需的时间,用PBS洗涤细胞单层一次
    13. 在室温下用稀释的200μlPBS/4%PFA固定10分钟
    14. 吸出固定剂和孵育5分钟在200微升PBS /甘氨酸淬灭PFA。
    15. (任选的)通过在20分钟内孵育细胞,可以在PBS中进行DAPI染色(0.2μg/ml)
    16. 用PBS洗涤3次。
    17. (任选的)如果需要,可以使细胞透化以根据测定特异性方案进行免疫荧光标记。我们常规使用0.1%Triton X-100 10分钟来透化细胞,同时保持GFP内在荧光。皂苷也可用于本申请,而基于甲醇/丙酮的方法不是。在PBS/1%BSA/0.2%明胶中进行封闭。然后应使用标准免疫荧光方法染色盖玻片。
      注意,在这种情况下,如上所述,在与第二抗体孵育期间进行DAPI染色。
    18. 用一对细镊子取出盖玻片,短暂浸没在蒸馏水中,用吸水纸除去多余的水。
    19. 使用10μl预热(37°C)封固剂在载玻片上安装。
    20. 在室温下干燥,不干扰CS至少一小时。
    21. 储存在4°C,直到准备观察。
    22. 使用GFP或FITC滤光片或用氩激光器的488nm波段可视化细菌中的GFP信号。 使用CFP滤波器或氩激光器的458nm波段可视化Cerulean信号。 mCherry使用Cy3滤光片或568nm激光显现。 我们不建议在显微镜应用中使用DsRed与GFP联用

  3. 活体显微镜
    1. 如果要平行测试​​几个条件,则TC7细胞在实验前48小时在常规生长培养基中以所需密度在具有玻璃底的35mm培养皿中或在8孔微量培养板中通过。 注意:我们还使用HeLa和LLC-MK2细胞。
      含有pTSAR1Ud2.4s的细菌的细菌悬浮液如上制备,并且常规使用聚赖氨酸包被的细菌(参见步骤B8),特别是监测进入,因为通过该预处理大大增强了进入效率。也可以通过小心地将培养皿或微板固定到离心机的板支架上来进行离心诱导的感染,而不用聚赖氨酸预涂细菌(参见步骤B3-8)。
      注意:可以通过用2μM细胞松弛素D预处理细胞来阻断聚赖氨酸包被的细菌的进入,同时保持对宿主细胞的粘附。然后可以在约20分钟内通过吸出含有细胞松弛素D的培养基,洗涤细胞单层一次并用活的成像培养基替换培养基在HeLa细胞上触发输入。最后一步可以在显微镜载物台上进行,以便监测导致进入的早期事件。
    2. 如所述(参见步骤B9-11),通过根据需要调节孵育时间,进行细菌感染的进入和追踪
    3. (可选)感染细胞的质膜可以在使用Cell Mask Deep Red的显微镜观察之前立即标记。染色通过将细胞与含有1:1,000稀释的细胞掩模的DMEM-HEPES一起温育5分钟来进行。染色的细胞用DMEM-HEPES洗涤三次,并在成像之前进行步骤C4
    4. 用2ml活成像培养基或PBS洗涤细胞单层一次,吸出,并向细胞单层中加入2ml活成像培养基。
    5. 将感染的细胞转移到显微镜的阶段,其理想地应该配备有受控的气氛室(例如37℃和5%CO 2)。
    6. 要跟踪细菌,在快速采集模式下(例如,使用2 x 2像素合并,可以减少采集时间),每分钟采集至少0.5-1幅图像。 GFP,mCherry和细胞膜深红可以分别在使用Cy3和Cy5过滤器的大多数显微镜系统上同时成像。

  4. 制备用于流式细胞术(FC)分析的样品
    1. 如上所述通过TC7细胞,但使用每个条件或动力学点要研究的6孔板的一到两个孔。根据实验所需的汇合水平,每孔分配1×10 5 -5 -2×10 5个细胞,考虑更高的汇合将有利于细胞 - 细胞扩散,但减少细菌的初始进入
    2. 如上所述生长细菌。携带pTSAR 1.3的菌株对于流式细胞术(FC)是理想的,因为来自核糖体rpsM - 启动子的DsRed MS165的组成型表达允许跟踪非分泌细菌。
      注意:在本协议中,我们建议使用聚赖氨酸包被的S. flexneri有利于进入和最大限度地恢复细菌的FC分析(见步骤B8)。
    3. TC7细胞用DMEM-HEPES(RT)洗涤两次
    4. 将2ml DMEM-HEPES细菌悬浮液加入含有TC7细胞的24孔板的每个孔中。
    5. 感染的诱导如上所述进行(参见步骤B3-8)
    6. 允许感染进行所需的时间(参见步骤B9-11)。
    7. 通过如(Aussel等人,2011)中所述的洗涤剂裂解,固定和离心,从感染的细胞回收细菌。简言之,将细胞在PBS中洗涤一次,在220μl中裂解 PBS/0.1%Triton X-100,从板上刮下,并通过加入440μlPBS/3%PFA固定60分钟。然后将细菌转移到1.5ml管中并通过两个离心步骤回收:200×g离心5分钟以沉淀细胞核;回收上清液而不干扰沉淀并在16,000×g离心上清液10分钟。
    8. 用50μlPBS/100mM甘氨酸回收沉淀。
    9. 在室温下10分钟后,样品在PBS中以1:10进一步稀释
    10. (可选)在这个阶段的样品可以保持在4℃,避光,至少三个星期
    11. 将样品转移到FC管中,并可进一步稀释,以便每秒记录约1,000-3,000个事件
    12. 为了调整补偿和其它FC参数,实际上使用用于感染的细菌培养物的稀释液和如上所述预先用PFA固定的未标记和单标记的菌株。

笔记

  1. 结果通常是高度可重复的。 然而,为了确保分泌细菌与非分泌细菌的比例在检查后的攻击后的给定时间是恒定的,因此建议确保受感染细胞在类似的汇合水平进行研究,因为当细菌不能 有效地进行细胞到细胞的扩散,不移动且因此不分泌细菌的比例增加。 类似地,感染前的细菌传代必须在高度控制的条件下进行,例如通过在每次重复实验时注意停止在相似OD 600的孵育。

食谱

  1. 10x磷酸盐缓冲盐水(PBS)
    将80g的NaCl与2g的KCl,14.4g的Na 2 HPO 4,2.4g的KH 2 PO 4
    用NaOH调节pH至7.4 将蒸馏的H 2 O加到1000ml
    中 过滤或高压灭菌
    存储在RT
    对于所有应用程序,使用1x解决方案
  2. PBS /10μg/ml聚赖氨酸
    PBS
    1:1000的聚赖氨酸储备溶液(10mg/ml,在水中,过滤灭菌并储存在-20℃)
  3. DMEM-HEPES
    DMEM
    20 mM HEPES
  4. 追逐媒体
    DMEM
    5%FBS
    50μg/ml庆大霉素
  5. 实时成像介质
    DMEM gfp -2
    5%FBS
    50μg/ml庆大霉素
    注意:Ringer的缓冲区可用于代替实时成像介质,特别是感染持续时间短。
  6. PBS/4%PFA
    10ml PFA(32%)
    8ml 10x PBS
    将蒸馏的H 2 O加至80ml
    等分并储存在-20°C
    在4℃下一周后丢弃等分试样
  7. PBS/100mM甘氨酸 0.375g甘氨酸 5ml 10×PBS
    将H <2> O加入到50ml ml/h 过滤灭菌(0.2μm)
    储存在4°C
  8. 5,000x DAPI储液
    1ml H 2 O 2 / 1 mg DAPI
  9. 安装介质
    注意:食谱来自罗切斯特大学医学中心,Michael Mastrangelo,Eric Yehling; 详情请参阅 http://www.urmc.rochester.edu/共焦 - 常规显微镜/文件/Mowiol52810.pdf。
    24克分析级甘油
    9.6g Mowiol 4-88
    24ml蒸馏的H 2 O 2 / 48ml 0.2M Tris缓冲液(pH8.5) 溶解完全将溶液装入500ml烧瓶中,在60-70℃的加热板上搅拌4-5小时 以每50ml锥体20-40ml等分试样
    在5,000xg离心15分钟以除去未溶解的残余物并小心地回收上清液
    添加2.5%的1,4-二氮杂双环[2,2,2] - 辛烷(DABCO)作为抗漂白剂
    在15或50ml锥形瓶中等分
    储存于-20°C长期储存。
  10. TC7细胞的生长培养基
    DMEM
    20%FCS
    1×青霉素/链霉素 1x非必需氨基酸

致谢

我们感谢Claude Parsot对稿件的意见。 FXCV是CIHR,EMBO,Marie-Curie-IRG和FRM研究员。 PS是EMBO和Marie Curie-IEF研究员,PJS是HHMI高级学者。这项工作是由ERC(PJS高级奖学金,232798号支持)。

参考文献

  1. Aussel,L.,Zhao,W.,Hebrard,M.,Guilhon,A.A.Viala,J.P.,Henri,S.,Chasson,L.,Gorvel,J.P.,Barras,F.and Meresse, 沙门氏菌解毒酶足以应对宿主的氧化爆发。/a> Mol Microbiol 80(3):628-640
  2. Campbell-Valois,F.X.,Schnupf,P.,Nigro,G.,Sachse,M.,Sansonetti,P.J.and Parsot,C.(2014)。 荧光记者揭示了志贺氏菌III型分泌物的开/关调节进入和细胞到细胞扩散期间的装置。 细胞宿主微生物 15(2):177-189。
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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。
Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Campbell-Valois, F. X., Schnupf, P. and Sansonetti, P. J. (2014). Design of a Transcription-based Secretion Activity Reporter (TSAR) for the Type III Secretion Apparatus of Shigella flexneri and Uses Thereof. Bio-protocol 4(20): e1270. DOI: 10.21769/BioProtoc.1270.
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F-X Campbell-Valois
Institut Pasteur
Citation from the protocol:
"Fixed samples microscopy
36-48 h before the experiment, detach a confluent TC7 cells monolayer with 0.25% trypsin/EDTA and distribute TC7 cells on coverslips no. 1.5 placed in wells of a 24-well plate in their normal growth medium. To follow entry events, seed 5 x 104 cells/well (approximately 50-75% confluence the day of the experiment). To favor cell-to-cell spread during longer infection times, seed 1 x 105 cells/well. (Optional) Fibronectin-coated coverslips can be used to permit faster attachment of the cells, but in this case, the number of cells seeded must be reduced about 25-50% to obtain confluence similar to what is described for non-coated coverslips.
The day before the experiment, a CR–positive colony harboring pTSAR1ud2.1 or pTSAR1Ud2.4s is picked from a CR-containing TCS ampicillin plate and used to inoculate one tube of 8 ml TCS broth supplemented with Amp and incubated overnight at 30 °C with shaking.
Notes:
CR induces T3SA activation and effector secretion; colonies with an active T3SA therefore display a red center where CR has accumulated while bacteria that have lost the virulence plasmid show up as larger white colonies (Figure 2).
Strains harboring pTSAR1Ud1.1 are used in place of pTSAR1Ud2.4 for multiple labeling experiments because it allows the use of the red laser/Cy3 filter for immunofluorescence."

In the second to last line of this paragraph, there is a typo; pTSAR1Ud1.1 should be read pTSAR1Ud2.1.
3/2/2015 12:40:36 AM Reply
Francois Campbell-Valois
University of Ottawa

I confirm this mistake. In the second to last line of this paragraph, there is a typo; pTSAR1Ud1.1 should be read pTSAR1Ud2.1.

3/2/2015 1:52:07 AM


Bio-protocol team Bio-protocol team
bio-protocol

Dear Dr. Campbell-Valois:

Thank you again for pointing out. Per your permission, we have changed "pTSAR1Ud1.1" to "pTSAR1Ud2.1" in the second to last line of that paragraph.

Sincerely,
Bio-protocol team

3/4/2015 10:27:01 PM