电功能性细菌效应器到哺乳动物细胞
Summary
Electroporation was used to insert purified bacterial virulence effector proteins directly into living eukaryotic cells. Protein localization was monitored by confocal immunofluorescence microscopy. This method allows for studies on trafficking, function, and protein-protein interactions using active exogenous proteins, avoiding the need for heterologous expression in eukaryotic cells.
Abstract
蛋白质相互作用的活细胞中的上下文中的研究可以产生大约本地化，动力学和相互作用伙伴的关键信息。这些信息是在宿主 – 病原体相互作用的背景下尤为可贵。许多病原体的蛋白质的宿主细胞内的多种方式，如功能，从而使宿主的免疫系统和存活的逃避细胞内环境中。研究这些病原体蛋白宿主-细胞相互作用，几种方法是常用的，其中包括： 在体内感染用表达一个标记或突变蛋白，或引入病原体的基因通过转染或转导的菌株。每一种方法都有优点和缺点。我们试图直接引入外源蛋白进入细胞的一种手段。电穿孔通常用于将核酸引入细胞中，但已被更很少应用于蛋白质虽然生物物理基础是完全一样的。一个标准的电穿孔被用于引入亲和标签效应细菌到哺乳动物细胞。人类上皮细胞和小鼠巨噬细胞用传统方法进行培养，分离，并置于0.4厘米的空隙电试管与感兴趣的外源细菌病原体蛋白（ 例如鼠伤寒沙门氏菌 GtgE）。电穿孔（0.3千伏）和短（4小时）的恢复期后，细胞内蛋白质是由通过其亲和标记物的荧光标记的蛋白，并检查空间和时间分布通过共聚焦显微镜证实。电穿孔的蛋白质也被证明是功能性细胞内，并能正确的亚细胞贩卖和蛋白质 – 蛋白质相互作用的。而该外源蛋白倾向于积聚在细胞的表面上，将电穿孔样品具有大量增加的细胞内效应器浓度相对单独孵育。该协议是简单，速度不够快，在做AParallel时尚，允许病原体蛋白在宿主细胞中的高通量特征包括亚细胞靶向和毒性蛋白的功能。
Introduction
许多革兰氏阴性菌采用专门的分泌系统注入毒性相关蛋白（称为效应器）直接导入宿主细胞1-5。这些效应物具有广泛的生物学功能，包括：抑制宿主免疫的，细胞骨架的变化，细胞内运输和信号传导，转录变化的修饰，和宿主蛋白酶的改变6-9。某些效应的功能是已知的，但是在主机的目标和其他许多人的生化作用（多个）仍有待确定。而比较野生型和重组体细菌感染是研究细胞内效应器毒力机制的有效的方法，它通常是有利的引入个体效应入宿主细胞。因此，简单的方法，用于在宿主细胞中的上下文中引入和表征细菌效应蛋白是高度期望的。
简化了实验分析的Wi第一个效应是至关重要的其它效应可能有相反的或多余的功能。要做到这一点简化，研究人员先前推出的大分子物质进入细胞的许多不同的方法，包括病毒转导10，显微注射11，刮装载12,13，细胞融合与化学诱导注射14，专有蛋白“转”试剂15，磷酸钙沉淀16，和电穿孔17-20。所引入的分子范围从核酸包括DNA，RNA和RNA干扰物种的蛋白质，细胞不透染料和抗体的细胞内目标21,22。一些方法都有局限性，包括大分子的可以引入的类型，以及特定下游分析可由于高的细胞毒性，作用破坏机制，低功效，或导入效率的限制。转染，一个ofte正使用的表达方法在哺乳动物细胞中的细菌的基因，也受到限制，一些有关的宿主细胞类型，例如巨噬细胞和原代细胞，是朝着转染特别耐。除此之外，它难以控制导入外源DNA后产生的细菌蛋白的水平。
许多工作已建立的核酸引入细菌和哺乳动物细胞作为通用实验室技术电穿孔;然而，人们一直在研究转化为在生理条件下提供的蛋白质进入细胞的最佳方法。对蛋白质的转染报告是有希望的，但需要昂贵的试剂和优化。引入潜在的有毒细菌效应成各种各样的以最小的成本细胞靶的愿望促使我们考虑电穿孔作为用于体内研究这些蛋白质的方法。
蛋白质电23-25 是一个满足HOD引入蛋白质成经由electropermeabilization活细胞，也被称为电-转染或电注入26。这种技术使用高强度的电脉冲以在细胞膜孔。这些可逆孔隙允许通常由细胞内的空间中排除大分子进入细胞。在去除外部电场，膜可以重新密封，使细胞能留存穿过孔27,28的分子。
一个标准的电穿孔被用于本研究中一致地引入细菌效应入两个小鼠巨噬细胞样细胞和人上皮细胞。该方法是快速的，有效的，廉价的，在细胞生存力没有明显的下降。引入的蛋白可通过免疫荧光显微镜进行可视化，或用于功能测定法。这已被证明使用绿色荧光蛋白（GFP），为无毒的标准，以及2 沙门氏菌效应蛋白，SspH1和GtgE。我们建议蛋白电作为剧目的细菌毒力的蛋白质及其功能的真核宿主细胞研究的一个额外的工具。
Protocol
Representative Results
Discussion
从致病细菌分泌的效应已经发展到在宿主细胞内环境的作用，因此它是有帮助的主机内研究它们在原位 。引入感兴趣的特定效应物到宿主细胞中允许相关病原体 – 宿主相互作用进行研究孤立，而不与其他细菌蛋白质的干扰。的目标是探讨电穿孔以引入细菌效应蛋白进入真核宿主细胞，从而避免了与转染或转导相关的挑战的一种手段。绿色荧光蛋白作为对照和沙门氏菌效应蛋白进行了…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported by NIGMS, National Institutes of Health (GM094623). Significant portions of this work were performed in the Environmental Molecular Sciences Laboratory, a DOE/BER national scientific user facility located at the Pacific Northwest National Laboratory (PNNL). PNNL is operated for the DOE by Battelle under Contract DE-AC05-76RLO1830.
Materials
| Material or Equipment | Company | Catalog Number | Comments |
| 0.25% Trypsin-EDTA Solution | Cellgro | 25-050-Cl | |
| 0.4cm Gap-disposable electroporation cuvettes | Bio-Rad | 165-2088 | |
| 100% Methanol | Any | N/A | Flammable, Toxic |
| Bovine Serum Albumin (BSA) | Sigma-Aldrich | A4919 | |
| Cell Counting Apparatus – Hemocytometer or Coulter Counter | Beckman Coulter | Model Z1 | |
| Cell Culture Incubator | Any | N/A | Humidified 95% air/5% CO2 atmosphere at 37 °C |
| Cell Culture Plastic | Any | N/A | Cell culture flasks/plates, pipets, tubes, rubber policeman |
| Dulbecco's Modification of Eagle’s Medium (DMEM) | Cellgro | 10-013 | Warm to 37 °C |
| Electroporator | Bio-Rad | E. coli Pulser | |
| Fetal Bovine Serum (FBS) | Cellgro | 35-016-CV | |
| Fluorescent confocal microscope | Ziess | Model LSM 710 | |
| Glass Bottom Dishes for Microscopy | Wilco Wells | HBSt-3522 | |
| HALT Protease Inhibitor Cocktail | Pierce | 78430 | Corrosive, Toxic |
| HeLa Cell Line | ATCC | ATCC CCL-2 | |
| LDS 4X Loading Buffer | Invitrogen | NP0007 | |
| Minimal Essential Medium (MEM) | Cellgro | 10-010 | Warm to 37 °C |
| Other fluorescent stains (WGA, DAPI) in conjunction with anti-fade reagent | Any | N/A | |
| Penicillin/Streptomycin | Cellgro | 30-002-Cl | |
| RAW 264.7 Cell line | ATCC | TIB-71 | |
| Primary Antibody Against Target of Interest | Any | N/A | |
| Secondary Antibody Conjugated to Fluorophore | Any | N/A | |
| Phosphate Buffered Saline | Any | N/A | Chill to 4 °C |
| Sterile Phosphate Buffered Saline | Any | N/A | Warm to 37 °C |
| Streptavidin Agarose Resin Suspension | Pierce | 20353 | |
| Table Top Centrifuge Capable of Accepting Conical Tubes (swinging bucket preferred) | Any | N/A | |
| TCEP | Sigma-Aldrich | 646547 | Corrosive, Toxic |
| Triton X-100 | Sigma-Aldrich | T8585 | Irritant, Toxic |
References
- Mota, L. J., Cornelis, G. R. The bacterial injection kit: type III secretion systems. Annals of Medicine. 37, 234-249 (2005).
- Galan, J. E., Collmer, A. Type III secretion machines: bacterial devices for protein delivery into host cells. Science. 284, 1322-1328 (1999).
- Hueck, C. J. Type III protein secretion systems in bacterial pathogens of animals and plants. Microbiology and molecular biology reviews : MMBR. 62, 379-433 (1998).
- Cornelis, G. R., Van Gijsegem, F. Assembly and function of type III secretory systems. Annual Review of Microbiology. 54, 735-774 (2000).
- He, S. Y. Type III protein secretion systems in plant and animal pathogenic bacteria. Annual Review of Phytopathology. 36, 363-392 (1998).
- Dean, P. Functional domains and motifs of bacterial type III effector proteins and their roles in infection. FEMS Microbiology Reviews. 35, 1100-1125 (2011).
- Espinosa, A., Alfano, J. R. Disabling surveillance: bacterial type III secretion system effectors that suppress innate immunity. Cellular Microbiology. 6, 1027-1040 (2004).
- Orchard, R. C., Alto, N. M. Mimicking GEFs: a common theme for bacterial pathogens. Cellular Microbiology. 14, 10-18 (2012).
- Galan, J. E. Common themes in the design and function of bacterial effectors. Cell Host Microbe. 5, 571-579 (2009).
- Ellis, B. L., et al. A survey of ex vivo/in vitro transduction efficiency of mammalian primary cells and cell lines with Nine natural adeno-associated virus (AAV1-9) and one engineered adeno-associated virus serotype. Virology Journal. 10, 74 (2013).
- Sreelatha, A., et al. Vibrio effector protein, VopQ, forms a lysosomal gated channel that disrupts host ion homeostasis and autophagic flux. Proceedings of the National Academy of Sciences of the United States of America. 110, 11559-11564 (2013).
- McNeil, P. L., Murphy, R. F., Lanni, F., Taylor, D. L. A method for incorporating macromolecules into adherent cells. The Journal of Cell Biology. 98, 1556-1564 (1984).
- Lafon, M., Lafage, M. Antiviral activity of monoclonal antibodies specific for the internal proteins N and NS of rabies virus. The Journal of General Virology. 68 (Pt. 12), 3113-3123 (1987).
- Kriegler, M. P., Livingston, D. M. Chemically facilitated microinjection of proteins into intact monolayers of tissue culture cells). Somatic Cell Genetics. 3, 603-610 (1977).
- Nossa, C. W., et al. Activation of the abundant nuclear factor poly(ADP-ribose) polymerase-1 by Helicobacter pylori. Proceedings of the National Academy of Sciences of the United States of America. 106, 19998-20003 (2009).
- Jordan, M., Schallhorn, A., Wurm, F. M. Transfecting mammalian cells: Optimization of critical parameters affecting calcium-phosphate precipitate formation. Nucleic Acids Research. 24, 596-601 (1996).
- Winegar, R. A., Phillips, J. W., Youngblom, J. H., Morgan, W. F. Cell Electroporation Is a Highly Efficient Method for Introducing Restriction Endonucleases into Cells. Mutation Research. 225, 49-53 (1989).
- Cortes, F., Ortiz, T. Chromosome damage induced by restriction endonucleases recognizing thymine-rich DNA sequences in electroporated CHO cells. International Journal of Radiation Biology. 61, 323-328 (1992).
- Cortes, F., Ortiz, T. Induction of chromosomal aberrations in the CHO mutant EM9 and its parental line AA8 by EcoRI restriction endonuclease: electroporation experiments. Mutation Research. 246, 221-226 (1991).
- Baron, S., Poast, J., Rizzo, D., McFarland, E., Kieff, E. Electroporation of antibodies, DNA, and other macromolecules into cells: a highly efficient method. Journal of Immunological Methods. 242, 115-126 (2000).
- Lewis, R. Electroporation edges toward clinic for both gene therapy and drug delivery. Genetic Engineering and Biotechnology News. 17, (1997).
- Kotzamanis, G., et al. CFTR expression from a BAC carrying the complete human gene and associated regulatory elements. Journal of Cellular and Molecular Medicine. 13, 2938-2948 (2009).
- Chakrabarti, R., Wylie, D. E., Schuster, S. M. Transfer of monoclonal antibodies into mammalian cells by electroporation. The Journal of Biological Chemistry. 264, 15494-15500 (1989).
- Graziadei, L., Burfeind, P., Bar-Sagi, D. Introduction of unlabeled proteins into living cells by electroporation and isolation of viable protein-loaded cells using dextran-fluorescein isothiocyanate as a marker for protein uptake. Analytical Biochemistry. 194, 198-203 (1991).
- Wilson, A. K., Horwitz, J., De Lanerolle, P. Evaluation of the electroinjection method for introducing proteins into living cells. The American Journal of Physiology. 260, C355-C363 (1991).
- Prasanna, G. L., Panda, T. Electroporation: Basic principles, practical considerations and applications in molecular biology. Bioprocess Engineering. 16, 261-264 (1997).
- Weaver, J. C., Chizmadzhev, Y. A. Theory of electroporation: A review. Bioelectrochemistry and Bioenergetics. 41, 135-160 (1996).
- Weaver, J. C. Electroporation theory. Concepts and mechanisms. Methods in Molecular Biology. 55, 3-28 (1995).
- McAteer, J. A., Douglas, W. H. Monolayer culture techniques. Methods in Enzymology. 58, 132-140 (1979).
- Ho, T. D., et al. Identification of GtgE, a novel virulence factor encoded on the Gifsy-2 bacteriophage of Salmonella enterica serovar Typhimurium. Journal of Bacteriology. 184, 5234-5239 (2002).
- Niemann, G. S., et al. Discovery of novel secreted virulence factors from Salmonella enterica serovar Typhimurium by proteomic analysis of culture supernatants. Infection and Immunity. 79, 33-43 (2011).
- Spano, S., Liu, X., Galan, J. E. Proteolytic targeting of Rab29 by an effector protein distinguishes the intracellular compartments of human-adapted and broad-host Salmonella. Proceedings of the National Academy of Sciences of the United States of America. 108, 18418-18423 (2011).
- Haraga, A., Miller, S. I. A Salmonella type III secretion effector interacts with the mammalian serine/threonine protein kinase PKN1. Cellular Microbiology. 8, 837-846 (2006).
- Zinchuk, V., Grossenbacher-Zinchuk, O., et al. Quantitative colocalization analysis of confocal fluorescence microscopy images. Current Protocols in Cell Biology / Editorial Board, Juan S. Bonifacino … [et al]. 4 (Unit 4.19), (2011).
- Kimple, M. E., Sondek, J., et al. Overview of affinity tags for protein purification. Current Protocols in Protein Science / Editorial Board, John E. Coligan … [et al]. 9 (Unit 9.9), (2004).
