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.

1. Generation of Stable Cell Lines Expressing the Protein of Interest
<ol>
	<li>Prepare media by adding heat-inactivated FCS and 1% Penicillin/Streptomycin to commercially available Dulbecco&#180;s Modified Eagle Medium (DMEM) supplemented with GlutaMAX-I, Pyruvate, and 4.5&#160;g/L D-Glucose.</li>
	<li>Cultivate HEK293 cells in media at 37&#160;&#176;C in 5%&#160;CO2. Sub-cultivate cells every third day. Remove the media and resuspend the cells in 15 ml fresh media. Pipette 1 ml into a new 75&#160;cm2 ...

The authors have nothing to disclose.

Many pathogenic bacteria harbor secretion systems to secrete or translocate bacterial effector proteins into the host cell. These effector proteins have the capacity to modulate processes and pathways in the host cell, allowing the bacteria to survive and replicate within their respective intracellular niche. Understanding the biochemical activities and the molecular mechanisms of the effector proteins will help towards a better understanding of pathogenicity and may help to develop new therapeutic tools to combat diseas...

The virulence of many Gram-negative bacterial pathogens depends on specialized secretion systems to hijack eukaryotic host cells. Bacteria use these secretion systems to inject bacterial virulence proteins (effectors) into the host cell to modulate a variety of cellular and biochemical activities. The study of effector proteins has not only provided remarkable insight into fundamental aspects of host/pathogen interactions but also into the basic biology of eukaryotic cells1. Modulation of host cell apoptosis has been shown to be an important virulence mechanism for many intracellular pathogens, and a number of effector proteins modulating apoptosis have been identified2-9. However, their precise molecular mechanisms of activity remain elusive in many cases.
Apoptosis, a form of programmed cell death, plays an important role in immune responses to infection10. Two main pathways leading to apoptosis have been identified: targeting the mitochondria (intrinsic apoptosis) or direct transduction of the signal via cell death receptors at the plasma membrane (extrinsic apoptosis). The intrinsic or mitochondria-mediated cell death pathway is triggered by intracellular signals and involves the activation of Bax and Bak, two pro-apoptotic members of the Bcl-2 family. This family is composed of pro- and anti-apoptotic regulator proteins that control cell death11-14. Activation of apoptosis leads to oligomerization of Bax and Bak followed by subsequent permeabilization of the mitochondrial outer membrane, resulting in cytochrome C release into the cytoplasm. Cytochrome C release initiates activation of the effector caspases 3 and 7 through activation of caspase 9 in the apoptosome15. This leads to proteolysis of selected substrates that, among others, results in the exposure of phosphatidylserine on the cell surface16 and frees a dedicated DNase that fragments chromatin17,18.
In order to determine where within the apoptotic cascade an individual effector protein interferes, an inducible expression system was employed19. Regulatory systems for conditional expression of transgenes have been an invaluable tool in analyzing a protein&#8217;s function within the cell or its importance for tissue, organ and organism development, as well as during initiation, progression and maintenance of disease20-23. Typically, inducible control systems, such as the Tet system24 employed here, form an artificial transcription unit (see Figure 1). One component is an artificially engineered transcription factor called tTA (tetracycline-dependent transcription activator), formed by fusion of the bacterial transcription repressor TetR25 to a mammalian protein domain that mediates transcriptional activation or silencing 24,26. The second component is a hybrid promoter, termed TRE (tetracycline-responsive element), consisting of a eukaryotic minimal promoter, containing at least a TATA-box and a transcription initiation site, joined to multiple repeats of the cognate DNA-binding site for TetR, tetO24,25. The third component is the natural ligand of TetR, tetracycline or one of its derivatives, such as anhydrotetracycline or doxycycline25. Upon ligand addition to the culture medium, TetR loses its affinity for tetO and dissociates from the TRE. As a result, transcription of the target gene is abolished. Transgene expression can, thus, be tightly controlled in a time- and dose-dependent manner in both cell culture and in animals20,23,24. With tTA, transgene expression occurs constitutively, except in the presence of a tetracycline. This can be a disadvantage in the study of cytotoxic or oncogenic proteins because tetracycline first has to be removed from the system, before transgene expression occurs and the target protein&#8216;s effects on the cell can be monitored. This can be time-consuming and is not always complete, especially in transgenic animals27. To address this limitation, a TetR mutant with an inverse response to the presence of doxycycline was used to generate a new transcription factor, rtTA (reverse tTA)28. It only binds to the TRE and, concomitantly, activates transcription in the presence of doxycycline. Residual leakiness of the system, i.e., transgene expression in the absence of TRE-bound transcription factor, originating either (i) from position effects at a genomic integration site, (ii) from the TRE itself29, or (iii) from non-specific binding of tTA/rtTA28, was addressed by introducing an additional transcriptional silencer, termed tTS (tetracycline-dependent transcriptional silencer)30 to the system. It forms a dual regulator network together with rtTA (see Figure 1). In the absence of doxycycline, tTS binds to TRE and actively shuts down any remaining transcription. In the presence of doxycycline, tTS dissociates from TRE and rtTA binds simultaneously inducing expression of the target gene. This additional layer of stringency is often necessary to express highly active cytotoxic proteins31-34.
Using this tightly controlled dual-regulator system, the apoptotic cascade can be initiated at a defined step allowing analysis of whether the given effector protein can interfere with apoptosis induction. This method can not only be used to study the anti-apoptotic activity of bacterial effector proteins but also for the inducible expression of pro-apoptotic or toxic proteins, or for dissecting interference with other signaling pathways.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>DMEM</td>
			<td>life technologies</td>
			<td>31966-021</td>
			<td></td>
		</tr>
		<tr>
			<td>FCS</td>
			<td>Biochrom</td>
			<td>S0115</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen/Strep</td>
			<td>life technologies</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>OptiMEM</td>
			<td>life technologies</td>
			<td>51985</td>
			<td></td>
		</tr>
		<tr>
			<td>X-tremeGENE 9</td>
			<td>Roche</td>
			<td>6365752001</td>
			<td></td>
		</tr>
		<tr>
			<td>Geneticin</td>
			<td>Roth</td>
			<td>CP11.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylenimine</td>
			<td>Polyscienes</td>
			<td>23966</td>
			<td></td>
		</tr>
		<tr>
			<td>Doxycycline</td>
			<td>Sigma Aldrich</td>
			<td>D9891</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-PROTEAN Tetra Cell</td>
			<td>Bio-Rad</td>
			<td>165-8000EDU</td>
			<td></td>
		</tr>
		<tr>
			<td>Trans-Blot SD Semi-Dry Transfer Cell</td>
			<td>Bio-Rad</td>
			<td>170-3940</td>
			<td></td>
		</tr>
		<tr>
			<td>PageRuler Prestained Protein Ladder</td>
			<td>Thermo Scientific</td>
			<td>26616</td>
			<td></td>
		</tr>
		<tr>
			<td>PVDF membrane</td>
			<td>Millipore</td>
			<td>IPVH00010</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-GFP&#160;</td>
			<td>life technologies</td>
			<td>A6455</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-cleaved PARP</td>
			<td>BD Bioscience</td>
			<td>611038</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-actin</td>
			<td>Sigma Aldrich</td>
			<td>A2066</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IgG (H+L)-HRPO</td>
			<td>Dianova</td>
			<td>111-035-062</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit IgG (H+L)-HRPO</td>
			<td>Dianova</td>
			<td>111-035-045</td>
			<td></td>
		</tr>
		<tr>
			<td>ECL Western Blotting Substrate</td>
			<td>Thermo Scientific</td>
			<td>32106</td>
			<td></td>
		</tr>
		<tr>
			<td>Restore Plus Western Blot Stripping Buffer</td>
			<td>Thermo Scientific</td>
			<td>46428</td>
			<td></td>
		</tr>
	</tbody>
</table>

applying an inducible expression system to study interference of bacterial virulence factors with intracellular signaling

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a signaling pathway. The method is based, on the one hand, on the inducible expression of a specific protein to initiate a signaling event at a defined and predetermined step in the selected signaling cascade. Concomitant expression, on the other hand, of the gene of interest then allows the investigator to evaluate if the activity of the expressed target protein is located upstream or downstream of the initiated signaling event, depending on the readout of the signaling pathway that is obtained. Here, the apoptotic cascade was selected as a defined signaling pathway to demonstrate protocol functionality. Pathogenic bacteria, such as Coxiella burnetii, translocate effector proteins that interfere with host cell death induction in the host cell to ensure bacterial survival in the cell and to promote their dissemination in the organism. The C. burnetii effector protein CaeB effectively inhibits host cell death after induction of apoptosis with UV-light or with staurosporine. To narrow down at which step CaeB interferes with the propagation of the apoptotic signal, selected proteins with well-characterized pro-apoptotic activity were expressed transiently in a doxycycline-inducible manner. If CaeB acts upstream of these proteins, apoptosis will proceed unhindered. If CaeB acts downstream, cell death will be inhibited. The test proteins selected were Bax, which acts at the level of the mitochondria, and caspase 3, which is the major executioner protease. CaeB interferes with cell death induced by Bax expression, but not by caspase 3 expression. CaeB, thus, interacts with the apoptotic cascade between these two proteins.

First, HEK293 cell lines stably expressing the protein of interest (CaeB) as a GFP-fusion protein were established. As a control, HEK293 cell lines stably expressing GFP were also generated. Expression of GFP and GFP-CaeB was verified by immunoblot analysis. The representative immunoblot (Figure 4A) demonstrates stable and clearly detectable expression of GFP and GFP-CaeB. However, this assay cannot determine whether all cells express GFP or GFP-CaeB. Therefore, the stably transfected HEK293 cell lines w...

Watch this Scientific Journal Video about Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling at JoVE.com

Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling

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.

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a ...

applying-an-inducible-expression-system-to-study-interference

Research

JoVE Journal

Immunology and Infection

9.1K ビュー.  Friedrich-Alexander-Universität. 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.