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Immunology and Infection

施加诱导表达系统研究与细胞内信号细菌毒力因子的干扰

Published: June 25, 2015
doi:
10.3791/52903
, , ,
1Department Biologie,Friedrich-Alexander-Universität, 2Institut für Molekulare Pathogenese,Friedrich-Loeffler-Institut, 3Mikrobiologisches Institut – Klinische Mikrobiologie, Immunologie und Hygiene,Universitätsklinikum Erlangen

Summary

The method described here is used to induce the apoptotic signaling cascade at defined steps in order to dissect the activity of an anti-apoptotic bacterial effector protein. This method can also be used for inducible expression of pro-apoptotic or toxic proteins, or for dissecting interference with other signaling pathways.

Abstract

这里提出的技术允许一个来分析在该步骤目标蛋白质，或者小分子，与信号传导途径的组分相互作用。该方法是基于，一方面，在一个特定的蛋白质的诱导表达引发的信令事件在所选择的信号级联限定和预定的步骤。相伴表达，另一方面，目的基因的则允许调查者评估，如果表达靶蛋白的活性被位于上游或发起信令事件的下游，根据所获得的信号通路的读出。这里，凋亡级联被选为一个定义信号传导途径来证明协议的功能。致病细菌，如贝氏柯克斯体 ，易位效应蛋白，与在宿主细胞宿主细胞死亡诱导干扰，以确保细菌存活的细胞，并促进其传播的生物体。 C.该贝氏柯克斯效应蛋白CAEB诱导细胞凋亡与UV光或具有星形孢菌素后有效抑制宿主细胞死亡。来缩小在该步骤CAEB干扰凋亡信号的传播，选择蛋白与充分表征的促凋亡活性在多西环素可诱导的方式瞬时表达。如果CAEB充当这些蛋白质的上游，将细胞凋亡进行畅通无阻。如果CAEB行为下游，细胞死亡会被抑制。选择的试验蛋白质分别为Bax蛋白，其作用在线粒体水平，和caspase 3，这是主要的刽子手蛋白酶。 CAEB干扰诱导Bax表达的细胞死亡，而不是由胱天蛋白酶3的表达。 CAEB，由此，交互这两种蛋白质之间的凋亡级联。

Introduction

许多革兰氏阴性细菌病原体的毒力取决于专门的分泌系统劫持真核宿主细胞。细菌利用这些分泌系统注入细菌毒性蛋白（效应物）到宿主细胞来调节各种细胞和生化活性。效应蛋白的研究不仅提供了卓越的洞察宿主/病原体相互作用的基本方面，但也到真核细胞1的基础生物学。宿主细胞凋亡的调节已被证明是一种重要的毒力机制的许多细胞内病原体，和一些效应蛋白调节细胞凋亡已经确定2-9。然而，他们的活动的精确的分子机制仍然是难以捉摸的，在许多情况下。

细胞凋亡，程序性细胞死亡的一种形式，在免疫应答中起重要作用，以感染10。导致细胞凋亡的两个主要途径有已经确定：针对线粒体（内在凋亡）或信号的直接转导通过细胞死亡受体在质膜（外源性凋亡）。本征或线粒体介导的细胞死亡途径由细胞内信号所触发的，并涉及Bax和Bak的，所述的Bcl-2家族的两个促凋亡成员的活化。这家由控制细胞死亡11-14亲和抗凋亡调节蛋白。细胞凋亡的激活导致低聚的Bax和Bak的后跟线粒体外膜的随后通透性，导致细胞色素C释放到细胞质中。细胞色素c释放引发的凋亡小15激活效应半胱氨酸蛋白酶3和7通过激活caspase 9的。这导致选择底物，除其他外，导致磷脂酰丝氨酸在细胞表面16的暴露的蛋白水解，并释放一个专用的DNase该片段沟道romatin 17,18。

为了确定在凋亡级联的个体效应蛋白干扰内，可诱导的表达系统被雇用19。管制系统，转基因的条件式已在分析过程中发生，发展和维持疾病20-23的细胞内的蛋白质的功能或它的对组织，器官和生物体发展的重要性，以及一个宝贵的工具。典型地，可诱导的控制系统，如四环素系统24在这里使用的，形成人工转录单位（参见图1）。一种成分是一种叫做tTA的（四环素依赖性转录激活剂）人工工程转录因子，通过融合的细菌转录阻遏四面体25到介导转录激活或沉默24,26的哺乳动物蛋白结构域形成。第二组分是一种混合启动子，称为TRE（四环素响应元件），它由一个真核最小启动子的，含有至少一个TATA盒和转录起始位点，结合到同源DNA结合位点为四面体，TETO 24,25的多个重复。第三成分是四面体，四环素或其衍生物，如无水四环素或强力霉素25之一的天然配体。在配体加入到培养基中，四面体失去其亲和力TETO并从TRE解离。其结果，靶基因的转录被废除。转基因表达可以，因此，被严格控制在两个细胞培养物以时间和剂量依赖的方式和在动物20,23,24。与tTA的，转基因的表达发生组成型，除了在四环素存在下进行。这可以是在细胞毒性或致癌蛋白的研究一个缺点，因为四环素首先必须从体系中除去，转基因expressio前n，将会和可监控单元上的靶蛋白的影响。这可能是耗时的并且不总是完全，特别是在转基因动物27。为了解决此限制，一个四面体突变体具有逆响应于多西环素的存在被用来产生新的转录因子，rtTA（反向TTA）28。它仅结合于TRE和，伴随，激活在强力霉素存在下转录。的系统， 即残余泄漏，在不存在TRE结合转录因子的转基因表达，始发或者（i）从位置效应在基因组整合位点，（ⅱ）从TRE本身29，或（iii）来自非特异性tTA的/ rtTA 28的结合，是通过引入一个附加的转录消音处理，称为TTS（四环素依赖的转录的消音器）30到系统。它形成了一个双重调节网络一起rtTA（ 见图1）。在没有强力霉素，TTS结合TRE积极关闭任何剩余的转录。在强力霉素存在时，TTS解离TRE和rtTA具约束力同时诱导靶基因的表达。严紧的这个附加层往往是必要的，表达高活性细胞毒性蛋白质31-34。

使用这种严格控制的双调节器系统，细胞凋亡级联可以在限定的步骤，使给定的效应蛋白是否能够与细胞凋亡诱导干扰分析来启动。这种方法不仅可用于研究细菌效应蛋白的抗凋亡活性，但也可用于促凋亡或有毒的蛋白质的诱导表达，或用于解剖与其他信号传导途径的干扰。

Protocol

1.产生的稳定细胞系的利益表达的蛋白通过加入热灭活的FCS和1％青霉素/链霉素准备媒体市售Dulbecco's改良的Eagle培养基（DMEM），补充有含有GlutaMAX-I，丙酮酸，和4.5g / L的D-葡萄糖。 在5％CO 2培养HEK293细胞中的媒体，在37℃。子培养细胞每三天。删除媒体和悬浮细胞15毫升新鲜培养基。吸管1毫升到一个新的75cm 2的细胞培养瓶中，并添加14毫升介质。 通过使用血…

Representative Results

首先，将HEK293细胞系稳定表达的兴趣（CAEB）的蛋白质作为GFP融合蛋白分别建立。作为对照，将HEK293细胞系稳定表达GFP的也产生。 GFP和GFP-CAEB的表达通过免疫印迹分析证实。代表性免疫印迹（ 图4A）显示GFP和GFP-CAEB的稳定和清晰地可检测的表达。然而，这种分析不能确定所有的细胞是否表达GFP或GFP-CAEB。因此，稳定地转染的HEK293细胞系还通过流式细胞仪分析。 如图4B所示 ，…

Discussion

许多病原细菌怀有分泌系统分泌或易位细菌效应蛋白进入宿主细胞。这些效应蛋白具有调节在宿主细胞中的过程和途径，从而使细菌存活并在其各自的细胞内利基复制的能力。了解生化活动和效应蛋白的分子机制将有助于建立一个更好地了解致病性，并可能有助于开发新的治疗手段，以抗击疾病。另外，作为效应蛋白经常通过模仿宿主细胞活动发挥它们的功能，它们也可以被用来进一步了解真核?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

This work was supported by the Deutsche Forschungsgemeinschaft (DFG) through the Collaborative Research Initiative 796 (SFB796) to A.L. and C.B., and through the ERA-NET PathoGenoMics 3rd call to A.L.

Materials

DMEM life technologies 31966-021
FCS Biochrom S0115
Pen/Strep life technologies 15140-122
OptiMEM life technologies 51985
X-tremeGENE 9 Roche 6365752001
Geneticin Roth CP11.3
Polyethylenimine Polyscienes 23966
Doxycycline Sigma Aldrich D9891
Mini-PROTEAN Tetra Cell Bio-Rad 165-8000EDU
Trans-Blot SD Semi-Dry Transfer Cell Bio-Rad 170-3940
PageRuler Prestained Protein Ladder Thermo Scientific 26616
PVDF membrane Millipore IPVH00010
anti-GFP  life technologies A6455
anti-cleaved PARP BD Bioscience 611038
anti-actin Sigma Aldrich A2066
Mouse IgG (H+L)-HRPO Dianova 111-035-062
Rabbit IgG (H+L)-HRPO Dianova 111-035-045
ECL Western Blotting Substrate Thermo Scientific 32106
Restore Plus Western Blot Stripping Buffer Thermo Scientific 46428
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References

  1. Galán, J. E. Common themes in the design and function of bacterial effectors. Cell Host Microbe. 5 (6), 571-579 (2009).
  2. Banga, S., et al. Legionella pneumophila inhibits macrophage apoptosis by targeting pro-death members of the Bcl2 protein family. Proc Natl Acad Sci USA. 104 (12), 5121-5126 (2007).
  3. Laguna, R. K., Creasey, E. A., Li, Z., Valtz, N., Isberg, R. R. A Legionella pneumophila-translocated substrate that is required for growth within macrophages and protection from host cell death. Proc Natl Acad Sci USA. 103 (49), 18745-18750 (2006).
  4. Schmid, M. C., et al. A translocated bacterial protein protects vascular endothelial cells from apoptosis. PLoS Pathog. 2 (11), e115 (2006).
  5. Nogueira, C. V., Carey, K. L., Roy, C. R. Inhibition of pathogen-induced apoptosis by a Coxiella burnetiitype IV effector protein. Proc Natl Acad Sci USA. 107 (44), 18997-19001 (2010).
  6. Rudel, T., Kepp, O., Kozjak-Pavlovic, V. Interactions between bacterial pathogens and mitochondrial cell death pathways. Nat Rev Microbiol. 8 (10), 693-705 (2010).
  7. Klingenbeck, L., Eckart, R. A., Berens, C., Lührmann, A. The Coxiella burnetii type IV secretion system substrate CaeB inhibits intrinsic apoptosis at the mitochondrial level. Cell Microbiol. 15 (4), 675-687 (2012).
  8. Raymond, B., et al. Subversion of trafficking, apoptosis, and innate immunity by type III secretion system effectors. Trends Microbiol. 21 (8), 430-441 (2013).
  9. Eckart, R. A., et al. Antiapoptotic activity of Coxiella burnetii effector protein AnkG is controlled by p32-dependent trafficking. Infect Immun. 82 (7), 2763-2771 (2014).
  10. Byrne, G. I., Ojcius, D. M. Chlamydia and apoptosis: life and death decisions of an intracellular pathogen. Nat Rev Microbiol. 2 (10), 802-808 (2004).
  11. Delft, M. F., Huang, D. C. S. How the Bcl-2 family of proteins interact to regulate apoptosis. Cell Res. 16 (2), 203-213 (2006).
  12. Willis, S. N., Adams, J. M. Life in the balance: how BH3-only proteins induce apoptosis. Curr Opin Cell Biol. 17 (6), 617-625 (2005).
  13. Shamas-Din, A., Kale, J., Leber, B., Andrews, D. W. Mechanisms of action of Bcl-2 family proteins. Cold Spring Harb Perspect Biol. 5 (4), a008714 (2013).
  14. Cory, S., Adams, J. M. The Bcl2 family: regulators of the cellular life-or-death switch. Nat Rev Cancer. 2 (9), 647-656 (2002).
  15. Adams, J. M., Cory, S. Apoptosomes: engines for caspase activation. Curr Opin Cell Biol. 14 (6), 715-720 (2002).
  16. Suzuki, J., Denning, D. P., Imanishi, E., Horvitz, H. R., Nagata, S. Xk-related protein 8 and CED-8 promote phosphatidylserine exposure in apoptotic cells. Science. 341 (6144), 403-406 (2013).
  17. Cory, S. Cell death throes. Proc Natl Acad Sci USA. 95 (21), 12077-12079 (1998).
  18. Enari, M., et al. A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD. Nature. 391 (6662), 43-50 (1998).
  19. Fussenegger, M. The impact of mammalian gene regulation concepts on functional genomic research, metabolic engineering, and advanced gene therapies. Biotechnol Prog. 17 (1), 1-51 (2001).
  20. Felsher, D. W. Reversibility of oncogene-induced cancer. Curr Opin Genet Dev. 14 (1), 37-42 (2004).
  21. Maherali, N., et al. A high-efficiency system for the generation and study of human induced pluripotent stem cells. Cell Stem Cell. 3 (3), 340-345 (2008).
  22. Kozlova, E. N., Berens, C. Guiding differentiation of stem cells in vivo by tetracycline-controlled expression of key transcription factors. Cell Transplant. 21 (12), 2537-2554 (2012).
  23. Reijmers, L., Mayford, M. Genetic control of active neural circuits. Front Mol Neurosci. 2, 27 (2009).
  24. Gossen, M., Bujard, H. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci USA. 89 (12), 5547-5551 (1992).
  25. Hillen, W., Berens, C. Mechanisms underlying expression of Tn10 .encoded tetracycline resistance. Annu Rev Microbiol. 48, 345-369 (1994).
  26. Deuschle, U., Meyer, W. K., Thiesen, H. J. Tetracycline-reversible silencing of eukaryotic promoters. Mol Cell Biol. 15 (4), 1907-1914 (1995).
  27. Robertson, A., Perea, J., Tolmachova, T., Thomas, P. K., Huxley, C. Effects of mouse strain, position of integration and tetracycline analogue on the tetracycline conditional system in transgenic mice. Gene. 282 (1-2), 65-74 (2002).
  28. Gossen, M., et al. Transcriptional activation by tetracyclines in mammalian cells. Science. 268 (5218), 1766-1769 (1995).
  29. Rang, A., Will, H. The tetracycline-responsive promoter contains functional interferon-inducible response elements. Nucleic Acids Res. 28 (5), 1120-1125 (2000).
  30. Freundlieb, S., Schirra-Müller, C., Bujard, H. A tetracycline controlled activation/repression system with increased potential for gene transfer into mammalian cells. J Gene Med. 1 (1), 4-12 (1999).
  31. Knott, A., et al. An optimized conditional suicide switch using doxycycline-dependent expression of human tBid. Cancer Biol Ther. 4 (5), 532-536 (2005).
  32. Danilchanka, O., et al. An outer membrane channel protein of Mycobacterium tuberculosis with exotoxin activity. Proc Natl Acad Sci USA. 111 (18), 6750-6755 (2014).
  33. Danke, C., et al. Adjusting transgene expression levels in lymphocytes with a set of inducible promoters. J Gene Med. 12 (6), 501-515 (2010).
  34. Maueröder, C., Chaurio, R. A., Platzer, S., Munoz, L. E., Berens, C. Model systems for rapid and slow induction of apoptosis obtained by inducible expression of pro-apoptotic proteins. Autoimmunity. 46 (5), 329-335 (2013).
  35. Srinivasula, S. M., et al. Generation of constitutively active recombinant caspases-3 and -6 by rearrangement of their subunits. J Biol Chem. 273 (17), 10107-10111 (1998).
  36. Baron, U., Freundlieb, S., Gossen, M., Bujard, H. Co-regulation of two gene activities by tetracycline via a bidirectional promoter. Nucleic Acids Res. 23 (17), 3605-3606 (1995).
  37. Anastassiadis, K., et al. A predictable ligand regulated expression strategy for stably integrated transgenes in mammalian cells in culture. Gene. 298 (2), 159-172 (2002).
  38. Qu, Z., et al. Homogeneity and long-term stability of tetracycline-regulated gene expression with low basal activity by using the rtTA2S-M2 transactivator and insulator-flanked reporter vectors. Gene. 327 (1), 61-73 (2004).
  39. Spencer, S. L., Gaudet, S., Albeck, J. G., Burke, J. M., Sorger, P. K. Non-genetic origins of cell-to-cell variability in TRAIL-induced apoptosis. Nature. 459 (7245), 428-432 (2009).
  40. Agha-Mohammadi, S., et al. Second-generation tetracycline-regulatable promoter: repositioned tet operator elements optimize transactivator synergy while shorter minimal promoter offers tight basal leakiness. J Gene Med. 6 (7), 817-828 (2004).
  41. Pluta, K., Luce, M. J., Bao, L., Agha-Mohammadi, S., Reiser, J. Tight control of transgene expression by lentivirus vectors containing second-generation tetracycline-responsive promoters. J Gene Med. 7 (6), 803-817 (2005).
  42. Hoffmann, A., Villalba, M., Journot, L., Spengler, D. A novel tetracycline-dependent expression vector with low basal expression and potent regulatory properties in various mammalian cell lines. Nucleic Acids Res. 25 (5), 1078-1079 (1997).
  43. Loew, R., Heinz, N., Hampf, M., Bujard, H., Gossen, M. Improved Tet-responsive promoters with minimized background expression. BMC Biotechnol. 10, 81 (2010).
  44. Heinz, N., et al. Retroviral and transposon-based tet-regulated all-in-one vectors with reduced background expression and improved dynamic range. Hum Gene Ther. 22 (2), 166-176 (2011).
  45. Toniatti, C., Bujard, H., Cortese, R., Ciliberto, G. Gene therapy progress and prospects: transcription regulatory systems. Gene Ther. 11 (8), 649-657 (2004).
  46. Ye, H., Fussenegger, M. Synthetic therapeutic gene circuits in mammalian cells. FEBS Lett. 588 (15), 2537-2544 (2014).

Tags

Inducible Expression SystemBacterial Virulence FactorsIntracellular SignalingApoptotic CascadeCoxiella BurnetiiCaeB Effector ProteinBaxCaspase 3Cell DeathSignaling PathwayProtein Interaction
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Berens, C., Bisle, S., Klingenbeck, L., Lührmann, A. Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling. J. Vis. Exp. (100), e52903, doi:10.3791/52903 (2015).
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