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Detection of the Secreted and Cytoplasmic Fractions of IpaB, IpaC and IpaD by Lysozyme Permeabilization
采用溶菌酶透化作用检测IpaB、IpaC和IpaD的分泌和细胞质切片   

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Abstract

Gram negative bacterial pathogens, such as Shigella flexneri, which possess a Type Three Secretion System (T3SS), are able to transfer bacterial proteins, dubbed translocators and effectors, from their cytoplasm into the cytoplasm of their host cells using a syringe like needle complex. For Shigella, it has been shown that during cellular invasion, the intrabacterial pool of translocators and effectors is completely depleted upon activation of the TTS Apparatus and is then progressively replenished while bacteria remain inside host cells. Replenishment of effectors allows for cell-to-cell spreading events, which also necessitate reactivation of the T3SA, and lead to another round of depletion of intrabacterial effector stores. To understand the state of individual intracellular bacteria during infection, it is therefore of interest to be able to locate and evaluate the relative quantity of the intrabacterial and secreted pool of translocators and effectors. We recently adapted a method based on EDTA and lysozyme to permeabilize the cell wall of bacteria present within host cells in order to label the intrabacterial pool of the tip protein IpaD and the translocators IpaB and IpaC. Herein, we describe in detail the protocol to perform the successive labeling of the intrabacterial and secreted pools. This method is theoretically extendable to virulence factors secreted by other secretion systems and other bacterial pathogens.

Materials and Reagents

  1. Shigella spp. Strain(s) (e.g. M90T)
  2. Human tissue culture cells such 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.
  3. Petri dish of Tryptone Casein Soja (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
  4. TCS broth (BD Biosciences, catalog number: 211825 )
  5. Polylysine (Sigma-Aldrich, catalog number: P1274 )
  6. DMEM (Life Technologies, catalog number: 31885 )
  7. FCS (Biowest, catalog number: S1810-100 )
  8. Penicillin/streptomycin (Life Technologies, catalog number: 15140 )
  9. Non-essential amino acids (Life Technologies, catalog number: 11140 )
  10. Trypsin-EDTA (Life Technologies, catalog number: 25200-056 )
  11. Fibronectin from human plasma (Sigma-Aldrich, catalog number: F0895 ) (optional)
  12. HEPES (Life Technologies, catalog number: 15630-056 )
  13. Gentamycin (EUROMEDEX, catalog number: EU0540-A )
  14. PFA (Electron Microscopy Sciences, catalog number: 15714 )
  15. Glycine (Sigma-Aldrich, catalog number: G7126 )
  16. Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
  17. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A 9647 )
  18. Gelatin (Sigma-Aldrich, catalog number: G1393 )
  19. Sodium azide (Sigma-Aldrich, catalog number: 0 8591 )
  20. Goat anti-mouse IgG Alexa568 (highly cross absorbed) (Life Technologies, catalog number: A11036 )
  21. Goat anti-mouse IgG Alexa647 (highly cross absorbed) (Life Technologies, catalog number: A21236 )
  22. Lysozyme (Sigma-Aldrich, catalog number: 6876 )
  23. 5, 000x DAPI stock solution (Sigma-Aldrich, catalog number: D9542 ) (optional) (see Recipes)
  24. MOWIOL (Sigma-Aldrich, catalog number: 81381 )
  25. 1,4-diazobicyclo-[2,2,2]-octane d (DABCO) (Sigma-Aldrich, catalog number: D27802 )
  26. Glycerol (VWR International, catalog number: 24388.295 ) alternatively Prolong mounting medium (Life Technologies, catalog number: P36930 )
  27. 10x phosphate-buffer saline (PBS) (see Recipes)
  28. PBS/10 µg/ml polylysine (see Recipes)
  29. DMEM/20 mM HEPES (see Recipes)
  30. Chase medium (see Recipes)
  31. PBS/4% PFA (see Recipes)
  32. PBS/100 mM glycine (see Recipes)
  33. PBS/0.1% Triton X-100 (see Recipes)
  34. PBS/1% BSA/0.2% gelatin (see Recipes)
  35. Lysozyme reaction buffer (see Recipes)
  36. 5,000x DAPI stock solution (see Recipes)
  37. Mounting medium (see Recipes)
  38. Growth medium for TC7 cells (see Recipes)

Equipment

  1. 24-well plates
  2. Coverslips no. 1.5, 12 mm in diameter (Harvard Apparatus, catalog number: 64-0712 )
  3. Laminar flow hood for cell culture
  4. Heating water bath
  5. Tabletop centrifuge for 1.5 ml Eppendorf tubes
  6. Tabletop centrifuge with plate-holding rotor
  7. CO2 incubator for cell culture
  8. Tweezers

Procedure

There are numerous studies in the literature describing protocols to perform immunofluorescence labeling of intrabacterial proteins (Buddelmeijer et al., 1998; Schlumberger et al., 2005). Although the detailed procedure might vary, they have in common the use EDTA and lysozyme, which destabilize the outer membrane [i.e. LPS, reviewed by Nikaido and Vaara (1985)] and degrade the peptidoglycan of the cell wall, respectively, hence allowing antibodies to diffuse into the bacterial cytoplasm to recognize their cognate antigen. Below we present the method that we have used to detect in combination the secreted and intrabacterial fractions of Shigella flexneri IpaB, IpaC and IpaD. See Reference 2 (Campbell-Valois et al., 2014) for application of the procedure.

  1. 36-48 h before the experiment, confluent TC7 cells monolayer (i.e. 100% coverage of the surface) are detached by incubation with 0.25 % trypsin-EDTA and distributed in their normal growth medium (see Recipes) onto sterile 12 mm coverslips (CS) no. 1.5 within wells of a 24-wells plate. 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 at longer infection times, seed 1 x 105 cells/well. (Optional) Fibronectin-coated coverslips (CS) can be used to favor attachment of the cells, but in this case, the number of cells seeded must be reduced approximately in half in order to obtain confluence similar to what is described for non-coated coverslips.
  2. Day before the experiment, a CR–positive S. flexneri colony (i.e. with a red dot at the center of the colony) is picked from a TCS-CR plate and used to inoculate a tube of TCS broth (e.g. 2-8 ml) for overnight growth at 30 °C with shaking.
    Note: 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 1).


    Figure 1. 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. (Optional) Permeabilization can be directly performed on bacteria grown in tubes. In this case, simply centrifuge 5 min at 450 x g a 1:5 dilution of the washed bacteria onto the required number of polylysine-coated 12 mm coverslips (incubate the sterile coverslips with 50 µg/ml polylysine in PBS for 60 min at RT, wash three times in 1 ml PBS); the use of polylysine-coated coverslips facilitates bacterial adhesion to the glass surface. Place coverslips in a 24-wells plate, wash two times with PBS and proceed with fixation, as described below.
  7. TC7 cells are washed two times with DMEM-HEPES (RT); after the washes place DMEM-HEPES at 37 °C for step 10.
  8. 500 µl of the DMEM-HEPES bacterial suspension is added to each well of the 24-wells plate containing the TC7 cells.
  9. 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 of non-coated bacteria. Polylysine-coated bacteria can be rapidly prepared by incubating bacteria 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.
  10. The bacteria suspension is aspirated and replaced by 500 µl of DMEM-HEPES preheated to 37 °C.
  11. Incubate between 15 to 30 min in a cell culture incubator with 5% CO2 at 37 °C.
  12. Aspirate and add the chase medium for the desired time. To study cell-to-cell spread in TC7 cells, a chase of 210 min is recommended.  
  13. At the desired time after infection, wash the cell monolayer once with PBS.
  14. Fix for 10 min with 200 µl PBS/4% PFA at room temperature (RT).
  15. Aspirate the fixative and incubate 5 min in 200 µl PBS/100 mM glycine to quench the PFA.
  16. Wash with 500 µl PBS 3 times.
  17. Incubate 4 min in 200 µl PBS/0.1% Triton X-100.
  18. Wash with 500 µl PBS 4 times.
  19. Incubate CS for 15 min in 200 µl blocking solution PBS/1% BSA/0.2% gelatin.
  20. Incubate CS ON at 4 °C with the primary antibody, directed either against IpaB, IpaC, IpaD or alternatively against any other secreted protein of interest, diluted in 200 µl blocking solution/coverslip. The dilution of the antibody is variable and must be determined empirically for each antibody.
  21. Wash 3 times with 500 µl PBS.
  22. Incubate for 120 min with the appropriate secondary antibody conjugated to Alexa Fluor 568 and diluted in blocking solution.
  23. Wash 3 times in 500 µl PBS.
  24. Incubate 5 min in 200 µl PBS/4% PFA to crosslink secondary and primary antibodies to their antigen and thus, avoid cross-reaction with the intrabacterial pool following lysozyme permeabilization.
  25. Incubate 5 min in 200 µl PBS/glycine.
  26. Wash 3 times in PBS.
  27. Incubate CS 20 min with the lysozyme reaction buffer at 37 °C.
  28. Wash 3 times in 500 µl PBS.
  29. Incubate overnight (ON) at 4 °C or 2-4 h at RT with the primary antibody directed against IpaB, IpaC, IpaD or alternatively against any other secreted proteins of interest diluted 200 µl blocking solution/coverslip.  Again, the dilution of the antibody is variable and must be determined empirically by the user.
  30. Wash 3 times in 500 µl PBS.
  31. Incubate for 120 min with the appropriate secondary antibody conjugated to Alexa Fluor 647 or equivalent fluorophore diluted 1/500-1/1,000 or according to supplier in 200 µl blocking solution/coverslip. At this step, DAPI can be added at 0.2 µg/ml during the incubation.
  32. Wash 3 times in PBS and heat mounting medium to 37 °C.
  33. Take out the CS from the plate with a fine pair of tweezers, briefly immerse in distilled water and remove excess water using a sheet of absorbing paper.
  34. Mount on a slide using 10 µl of pre-warmed (37 °C) mounting medium.
  35. Let dry without disturbing the CS for at least one hour at RT.
  36. Store at 4 °C until ready to observe.
  37. Expected results: Secreted fraction will appear in both the Alexa568 and Alexa647 channels, while the intrabacterial fraction will only appear in the Alexa647 channel. Localization of the secreted fraction will appear different than those of the intrabacterial fraction. Due to the sequential permeabilization procedure and cross-linking with PFA between labeling of the secreted and intrabacterial fractions, it is possible to use sequentially the same primary antibodies directed against the translocators or effectors to perform this protocol without compromising interpretation of the results  [see Figure 4C in Campbell-Valois et al. (2014) and Figure 2 below].  In most cases, bacteria with secreted translocators or tip proteins in their vicinity have depleted intrabacterial store. Sometime bacteria with no detectable intrabacterial or secreted store are observed. They most likely represent bacteria that have recently depleted their intrabacterial store and have not replenished their stock yet.


    Figure 2. Example of labeling obtained following the protocol. Here we show the final result obtained with the labeling protocol described above using a single monoclonal antibody (A8.1) directed against the tip protein paD expressed by Wt Shigella flexneri.  The arrow points at a bacterium, which has only intrabacterial IpaD protein (+lysozyme) and no detectable secreted fraction (-lysozyme). The large arrowhead points at a bacterium with secreted IpaD in its vicinity and demonstrate the cross-reaction described in step 37 that is systematically observed for bacteria with secreted IpaB, IpaC and IpaD in their vicinity, following lysozyme treatment. Contrary to bacteria similar to the one indicated by the arrow, the bacteria indicated by the large arrowhead do not have IpaD in its cytoplasm, as indicated by the hollow shape of the labeling.
    Note: Cerulean is a cyan variant of the green fluorescent protein from Aequoria victoria, here expressed from a constitutively active promoter by the bacteria.

Notes

  1. Results are usually highly reproducible. However to ensure that the ratio of secreting bacteria to not secreting bacteria be constant at a 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, 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. This is particularly important if the labelings are performed directly on bacteria grown in broth or at early time points post-challenge as the amount of translocators found in the bacterial cytoplasm is known to increase throughout the exponential growth phase.

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
    Sterilize by autoclaving
    Stored at RT
    Diluted to 1x concentration for all uses
  2. PBS/10 µg/ml polylysine
    1x PBS
    1:1,000 of the polylysine stock solution (10 mg/ml in water, filter sterilized and stored at -20 °C in 1 ml aliquots)
  3. DMEM/20 mM HEPES
    DMEM
    1:50 dilution of 1 M HEPES
  4. Chase medium
    DMEM
    5% FBS
    Gentamycin 50 µg/ml
  5. PBS/4% PFA
    10 ml PFA (32%)
    8 ml 10x PBS
    Add distilled H2O to 80 ml
    Aliquot and store at -20 °C
    Discard aliquot after one week at 4 °C
  6. PBS/100 mM glycine
    0.375 g glycine
    5 ml 10x PBS
    Add H2O to 50 ml
    Filter sterilize (0.2 µm)
    Stored at 4 °C
  7. PBS/0.1% Triton X-100
    500 µl PBS/10% Triton X-100 stock solution
    5 ml 10x PBS stock solution
    Filter sterilize (0.2 µm)
    Add distilled H2O to 50 ml
    Stored at 4 °C
  8. PBS/1% BSA/0.2% gelatin
    0.5 g BSA
    0.1 g gelatin
    50 ml 1x PBS
    Add sodium azide to final 2 mM
    Filter sterilize (0.2 µm)
    Stored at 4 °C
  9. Lysozyme reaction buffer
    PBS 0.8x from 10x PBS stock solution
    50 mM glucose
    5 mM EDTA
    Supplement immediately before use with 5 mg/ml lysozyme using a 100 mg/ml stock solution Aliquoted and stored at -20 °C
  10. 5,000x DAPI stock solution
    1 ml H2O
    1 mg DAPI
  11. 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 tube
    Centrifuge at 5,000 x g for 15 min to remove undissolved residues and carefully recover the supernatant
    Add 2.5% DABCO as anti-bleaching agent
    Aliquot in 15 or 50 ml conical tubes and stored at -20 °C for long-term storage
  12. 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). The procedure described above was originally described in Campbell-Valois et al. (2014).

References

  1. Buddelmeijer, N., Aarsman, M. E., Kolk, A. H., Vicente, M. and Nanninga, N. (1998). Localization of cell division protein FtsQ by immunofluorescence microscopy in dividing and nondividing cells of Escherichia coli. J Bacteriol 180(23): 6107-6116.
  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.
  3. Nikaido, H. and Vaara, M. (1985). Molecular basis of bacterial outer membrane permeability. Microbiol Rev 49(1): 1-32.
  4. Schlumberger, M. C., Müller, A. J., Ehrbar, K., Winnen, B., Duss, I., Stecher, B. and Hardt, W.-D. (2005). Real-time imaging of type III secretion: Salmonella SipA injection into host cells. Proc Natl Acad Sci U S A 102(35): 12548-12553.

简介

具有三型分泌系统(T3SS)的革兰氏阴性细菌病原体如灵芝氏菌能够将细菌蛋白质,配位的易位蛋白和效应子从其细胞质转移到其宿主细胞的细胞质中使用注射器如针复合物。对于志贺氏菌(Shigella),已经表明在细胞侵袭过程中,转位子和效应子的细菌池在TTS装置激活时完全耗尽,然后逐渐补充,同时细菌保留在宿主细胞内。补充效应物允许细胞间的扩散事件,这也需要T3SA的再活化,并导致细胞内效应子库的另一轮消耗。为了理解感染期间单个细胞内细菌的状态,因此有兴趣的是能够定位和评估细菌和分泌的易位蛋白和效应子池的相对数量。我们最近改编了基于EDTA和溶菌酶的方法以透化宿主细胞内存在的细菌的细胞壁,以标记尖端蛋白IpaD和易位蛋白IpaB和​​IpaC的细菌池。在这里,我们详细描述执行连续标记细菌和分泌池的协议。这种方法理论上可扩展到由其他分泌系统和其他细菌病原体分泌的毒力因子。

材料和试剂

  1. 志贺氏菌属。 应变(如 M90T)
  2. 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。
  3. 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佐剂(TCS)琼脂(BD Biosciences,目录号:236950)
  4. TCS肉汤(BD Biosciences,目录号:211825)
  5. 聚赖氨酸(Sigma-Aldrich,目录号:P1274)
  6. DMEM(Life Technologies,目录号:31885)
  7. FCS(Biowest,目录号:S1810-100)
  8. 青霉素/链霉素(Life Technologies,目录号:15140)
  9. 非必需氨基酸(Life Technologies,目录号:11140)
  10. 胰蛋白酶-EDTA(Life Technologies,目录号:25200-056)
  11. 来自人血浆的纤连蛋白(Sigma-Aldrich,目录号:F0895)(任选)
  12. HEPES(Life Technologies,目录号:15630-056)
  13. 庆大霉素(EUROMEDEX,目录号:EU0540-A)
  14. PFA(Electron Microscopy Sciences,目录号:15714)
  15. 甘氨酸(Sigma-Aldrich,目录号:G7126)
  16. Triton X-100(Sigma-Aldrich,目录号:T8787)
  17. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A 9647)
  18. 明胶(Sigma-Aldrich,目录号:G1393)
  19. 叠氮化钠(Sigma-Aldrich,目录号:08591)
  20. 山羊抗小鼠IgG Alexa568(高度交叉吸收)(Life Technologies,目录号:A11036)
  21. 山羊抗小鼠IgG Alexa647(高度交叉吸收)(Life Technologies,目录号:A21236)
  22. 溶菌酶(Sigma-Aldrich,目录号:6876)
  23. 5,000x DAPI储备溶液(Sigma-Aldrich,目录号:D9542)(可选)(参见配方)
  24. MOWIOL(Sigma-Aldrich,目录号:81381)
  25. 1,4-二偶氮双环 - [2,2,2] - 辛烷d(DABCO)(Sigma-Aldrich,目录号:D27802)
  26. 甘油(VWR International,目录号:24388.295)或长寿命封固剂(Life Technologies,目录号:P36930)
  27. 10x磷酸盐缓冲盐水(PBS)(参见配方)
  28. PBS /10μg/ml聚赖氨酸(见Recipes)
  29. DMEM/20mM HEPES(参见配方)
  30. Chase介质(见配方)
  31. PBS/4%PFA(参见配方)
  32. PBS/100mM甘氨酸(参见配方)
  33. PBS/0.1%Triton X-100(参见配方)
  34. PBS/1%BSA/0.2%明胶(见配方)
  35. 溶菌酶反应缓冲液(参见配方)
  36. 5,000x DAPI储备溶液(见配方)
  37. 安装介质(参见配方)
  38. TC7细胞的生长培养基(参见配方)

设备

  1. 24孔板
  2. Coverslips no。 1.5,直径12mm(Harvard Apparatus,目录号:64-0712)
  3. 用于细胞培养的层流罩
  4. 加热水浴
  5. 台式离心机用于1.5ml Eppendorf管
  6. 带托板转子的台式离心机
  7. CO 2细胞培养箱中培养
  8. 镊子

程序

在文献中有许多研究描述了对细菌内蛋白质进行免疫荧光标记的方案(Buddelmeijer等人,1998; Schlumberger等人,2005)。尽管详细的程序可以变化,但是它们通常使用EDTA和溶菌酶,其使外膜[即LPS,由Nikaido和Vaara(1985)综述]不稳定并降解细胞壁的肽聚糖,因此允许抗体扩散到细菌细胞质中以识别它们的同源抗原。下面我们提出我们已经用于组合检测灵芝志贺氏菌IpaB,IpaC和IpaD的分泌的和细菌内部分的方法。参见参考文献2(Campbell-Valois等人,2014),以应用该程序。

  1. 在实验前36-48小时,通过用0.25%胰蛋白酶-EDTA孵育分离汇合的TC7细胞单层(即,表面的100%覆盖),并分布在它们的正常生长培养基(参见Recipes)中无菌12mm盖玻片(CS) 1.5在24孔板的孔内。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)。为了在更长的感染时间促进细胞与细胞的铺展,以1×10 5个细胞/孔种子。 (任选的)纤连蛋白包被的盖玻片(CS)可用于促进细胞的附着,但是在这种情况下,接种的细胞数目必须减少大约一半,以获得类似于未涂布的盖玻片。
  2. 实验前一天,CR阳性。从TCS-CR板中挑取具有在菌落中心的红色菌落(即在菌落中心具有红色点)的菌落,并用于接种TCS肉汤的管(例如 em,2-8ml)在30℃振荡生长过夜。
    注意: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1)。


    图1.刚果红阳性(CR +)对刚果红阴性(CR-)集落将Wt和质粒固化的M90T衍生菌株在含琼脂的TCS和刚果红板上并排划线以显示其不同的表型。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. 第二天早晨,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℃孵育直到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. (可选)可以直接对管中生长的细菌进行透化。在这种情况下,简单地将洗涤细菌的1:5稀释液离心5分钟至所需数量的聚赖氨酸包被的12mm盖玻片上(将无菌盖玻片与50μg/ml聚赖氨酸一起孵育PBS在室温下60分钟,在1ml PBS中洗涤三次);使用聚赖氨酸包被的盖玻片有助于细菌粘附到玻璃表面。地点 盖玻片在24孔板中,用PBS洗涤两次并进行固定,如下所述
  7. TC7细胞用DMEM-HEPES(RT)洗涤两次;洗涤后,在37℃下将DMEM-HEPES置于步骤10
  8. 将500μlDMEM-HEPES细菌悬浮液加入含有TC7细胞的24孔板的每个孔中。
  9. 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未包被的细菌的离心。聚赖氨酸包被的细菌可以通过将孵育用1ml PBS(参见步骤4)在PBS /聚赖氨酸(10μg/ml)中洗涤一次的细菌10分钟并温和搅拌并在感染前洗涤细菌三次而快速制备。使用聚赖氨酸处理大大提高了进入效率,因此OD 600等于0.02-0.2的细菌悬浮液应与细胞在RT下温育10分钟,而不进行离心。
  10. 吸出细菌悬浮液并用预热至37℃的500μlDMEM-HEPES代替
  11. 在37℃下在具有5%CO 2的细胞培养箱中孵育15至30分钟。
  12. 吸出并添加追踪培养基所需的时间。 为了研究在TC7细胞中的细胞到细胞的传播,推荐210分钟的追踪。  
  13. 在感染后的所需时间,用PBS洗涤细胞单层一次
  14. 在室温(RT)下用200μlPBS/4%PFA固定10分钟
  15. 吸出固定剂,在200μlPBS/100mM甘氨酸中孵育5分钟以淬灭PFA。
  16. 用500μlPBS洗涤3次。
  17. 在200μlPBS/0.1%Triton X-100中孵育4分钟
  18. 用500μlPBS洗涤4次。
  19. 在200μl封闭溶液PBS/1%BSA/0.2%明胶中孵育CS 15分钟
  20. 孵育CS ON在4℃与第一抗体,针对IpaB,IpaC,IpaD或备选地对任何其他分泌的目标蛋白质,稀释在200μl封闭溶液/盖玻片。 抗体的稀释是可变的,并且必须对每种抗体凭经验确定。
  21. 用500μlPBS洗涤3次。
  22. 用与Alexa Fluor 568偶联的适当的二抗孵育120分钟,并在封闭溶液中稀释
  23. 在500μlPBS中洗涤3次。
  24. 在200μlPBS/4%PFA中孵育5分钟,以使第二抗体和第一抗体交联其抗原,从而避免溶菌酶透化后与细菌池的交叉反应。
  25. 在200μlPBS /甘氨酸孵育5分钟
  26. 在PBS中洗涤3次。
  27. 在37℃下用溶菌酶反应缓冲液孵育CS 20分钟
  28. 在500μlPBS中洗涤3次。
  29. 在4℃下孵育过夜(ON)或在室温下用针对IpaB,IpaC,IpaD的一级抗体或者针对稀释200μl封闭溶液/盖玻片的任何其它分泌的蛋白质孵育2-4小时。 同样,抗体的稀释是可变的,并且必须由用户凭经验确定。
  30. 在500μlPBS中洗涤3次。
  31. 孵育120分钟与适当的第二抗体偶联Alexa Fluor 647或稀释1/500-1/1,000等价荧光或根据供应商在200μl封闭溶液/盖玻片。 在此步骤中,DAPI可在孵育期间以0.2μg/ml添加
  32. 在PBS中洗涤3次,并将封固介质加热至37℃
  33. 用一对细镊子从培养皿中取出CS,短暂浸入蒸馏水中,用一张吸水纸除去多余的水。
  34. 使用10μl预热(37°C)封固剂在载玻片上安装。
  35. 让干燥,不要在室温下干扰CS至少一个小时。
  36. 储存在4°C,直到准备观察。
  37. 预期结果:分泌的部分将出现在Alexa568和Alexa647通道,而细菌内部分只会出现在Alexa647通道。分泌的部分的定位将看起来不同于细菌内部分的定位。由于在标记分泌的和细菌内部分之间的顺序渗透程序和与PFA的交联,可以顺序地使用针对易位蛋白或效应物的相同的一抗来实施该方案,而不损害结果的解释。参见图4C在Campbell-Valois等人(2014)和图2]。在大多数情况下,在其附近具有分泌的易位蛋白或末端蛋白的细菌具有耗尽的细菌存储。观察到有时没有可检测的细菌或分泌贮存的细菌。它们最可能代表最近消耗了细菌存储并且还没有补充其库存的细菌

    图2.根据方案获得的标记的实施例在这里,我们显示了使用针对由Wt表达的末端蛋白paD的单个单克隆抗体(A8.1)的上述标记方案获得的最终结果 Shigella flexneri 。箭头指向细菌,其仅具有细菌内IpaD蛋白(+溶菌酶)且没有可检测的分泌部分( - 溶菌酶)。大箭头指向在其附近具有分泌的IpaD的细菌,并且显示在步骤37中描述的交叉反应,其在溶菌酶处理后系统地观察到在其附近具有分泌的IpaB,IpaC和IpaD的细菌。与类似于箭头所示的细菌相反,由大箭头指示的细菌在其细胞质中不具有IpaD,如标记的中空形状所指示的。
    注意:Cerulean是来自Aequoria victoria的绿色荧光蛋白的青色变体,这里由细菌的组成型活性启动子表达。

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,建议确保感染的细胞在类似的汇合水平下进行研究,因为当细菌不能进行细胞细胞有效扩散,不移动,因此不分泌细菌的比例增加。类似地,感染前的细菌传代培养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。如果直接对在肉汤中生长的细菌或在攻击后的早期时间点进行标记,因为已知在细菌细胞质中发现的易位蛋白的量在整个指数生长期增加,这是特别重要的。

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 高压灭菌
    灭菌 存储在RT
    稀释至1倍浓度用于所有用途
  2. PBS /10μg/ml聚赖氨酸
    1x PBS
    1:1,000的聚赖氨酸储备溶液(10mg/ml,在水中,过滤灭菌并在-20℃下以1ml等分试样储存)
  3. DMEM/20mM HEPES
    DMEM
    1:50稀释的1 M HEPES
  4. 追逐媒体
    DMEM
    5%FBS
    庆大霉素50μg/ml
  5. PBS/4%PFA
    10ml PFA(32%)
    8ml 10x PBS
    将蒸馏的H 2 O加至80ml
    等分并存储在-20°C
    在4℃下一周后丢弃等分试样
  6. 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
  7. PBS/0.1%Triton X-100 500μlPBS/10%Triton X-100储备液
    5 ml 10x PBS储备液
    过滤灭菌(0.2μm)
    将蒸馏的H 2 O加入到50ml的
    中 储存在4°C
  8. PBS/1%BSA/0.2%明胶
    0.5 g BSA
    0.1克明胶
    50 ml 1x PBS
    加入叠氮化钠至最终2 mM
    过滤灭菌(0.2μm)
    储存在4°C
  9. 溶菌酶反应缓冲液
    PBS 0.8x从10x PBS储备液中洗涤
    50mM葡萄糖 5 mM EDTA
    在使用之前立即使用5mg/ml溶菌酶,使用100mg/ml储备溶液分装并储存在-20℃下
  10. 5,000x DAPI储液
    1ml H 2 O 2 / 1 mg DAPI
  11. 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) 为了溶解,将溶液完全装入500ml烧瓶中,在60-70℃的加热板上搅拌4-5小时
    在每50ml锥形管中以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
    加入2.5%DABCO作为抗漂白剂
    在15或50ml锥形管中等分,并储存在-20°C长期贮存
  12. TC7细胞的生长培养基
    DMEM
    20%FCS
    1×青霉素/链霉素 1个非必需氨基酸

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)。 上述程序最初在Campbell-Valois等人(2014)中描述。

参考文献

  1. Buddelmeijer,N.,Aarsman,M.E.,Kolk,A.H.,Vicente,M。和Nanninga,N。(1998)。 通过免疫荧光显微镜在细胞分裂和非分裂细胞中定位细胞分裂蛋白FtsQ,/em>。 180(23):6107-6116。
  2. Campbell-Valois,F.X.,Schnupf,P.,Nigro,G.,Sachse,M.,Sansonetti,P.J.and Parsot,C.(2014)。 荧光记者揭示志贺氏菌III型分泌器官在进入期间的开/关调节和细胞 - 细胞扩散 细胞宿主微生物 15(2):177-189
  3. Nikaido,H。和Vaara,M。(1985)。 细菌外膜通透性的分子基础 Microbiol Rev 49(1):1-32。
  4. Schlumberger,M.C.,Müller,A.J.,Ehrbar,K.,Winnen,B.,Duss,I.,Stecher,B.and Hardt,W.-D. (2005)。 III型分泌物的实时成像:沙门氏菌SipA注射入宿主细胞。 Proc Natl Acad Sci USA 102(35):12548-12553。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Campbell-Valois, F. X., Schnupf, P. and Sansonetti, P. J. (2014). Detection of the Secreted and Cytoplasmic Fractions of IpaB, IpaC and IpaD by Lysozyme Permeabilization. Bio-protocol 4(20): e1271. DOI: 10.21769/BioProtoc.1271.
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