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
发布: 2014年10月20日第4卷第20期 DOI: 10.21769/BioProtoc.1271 浏览次数: 10130
评审: Kanika GeraAnonymous reviewer(s)
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Feb 2014
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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
- Shigella spp. Strain(s) (e.g. M90T)
- 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. - 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
- TCS broth (BD Biosciences, catalog number: 211825 )
- Polylysine (Sigma-Aldrich, catalog number: P1274 )
- DMEM (Life Technologies, catalog number: 31885 )
- FCS (Biowest, catalog number: S1810-100 )
- Penicillin/streptomycin (Life Technologies, catalog number: 15140 )
- Non-essential amino acids (Life Technologies, catalog number: 11140 )
- Trypsin-EDTA (Life Technologies, catalog number: 25200-056 )
- Fibronectin from human plasma (Sigma-Aldrich, catalog number: F0895 ) (optional)
- HEPES (Life Technologies, catalog number: 15630-056 )
- Gentamycin (EUROMEDEX, catalog number: EU0540-A )
- PFA (Electron Microscopy Sciences, catalog number: 15714 )
- Glycine (Sigma-Aldrich, catalog number: G7126 )
- Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
- Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A 9647 )
- Gelatin (Sigma-Aldrich, catalog number: G1393 )
- Sodium azide (Sigma-Aldrich, catalog number: 0 8591 )
- Goat anti-mouse IgG Alexa568 (highly cross absorbed) (Life Technologies, catalog number: A11036 )
- Goat anti-mouse IgG Alexa647 (highly cross absorbed) (Life Technologies, catalog number: A21236 )
- Lysozyme (Sigma-Aldrich, catalog number: 6876 )
- 5, 000x DAPI stock solution (Sigma-Aldrich, catalog number: D9542 ) (optional) (see Recipes)
- MOWIOL (Sigma-Aldrich, catalog number: 81381 )
- 1,4-diazobicyclo-[2,2,2]-octane d (DABCO) (Sigma-Aldrich, catalog number: D27802 )
- Glycerol (VWR International, catalog number: 24388.295 ) alternatively Prolong mounting medium (Life Technologies, catalog number: P36930 )
- 10x phosphate-buffer saline (PBS) (see Recipes)
- PBS/10 µg/ml polylysine (see Recipes)
- DMEM/20 mM HEPES (see Recipes)
- Chase medium (see Recipes)
- PBS/4% PFA (see Recipes)
- PBS/100 mM glycine (see Recipes)
- PBS/0.1% Triton X-100 (see Recipes)
- PBS/1% BSA/0.2% gelatin (see Recipes)
- Lysozyme reaction buffer (see Recipes)
- 5,000x DAPI stock solution (see Recipes)
- Mounting medium (see Recipes)
- Growth medium for TC7 cells (see Recipes)
Equipment
- 24-well plates
- Coverslips no. 1.5, 12 mm in diameter (Harvard Apparatus, catalog number: 64-0712 )
- Laminar flow hood for cell culture
- Heating water bath
- Tabletop centrifuge for 1.5 ml Eppendorf tubes
- Tabletop centrifuge with plate-holding rotor
- CO2 incubator for cell culture
- Tweezers
Procedure
文章信息
版权信息
© 2014 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
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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分类
微生物学 > 微生物生物化学 > 蛋白质 > 分离和纯化
微生物学 > 微生物-宿主相互作用 > 细菌
生物化学 > 蛋白质 > 标记
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