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 cell culture flask and add 14 ml media.</li>
	<li>Analyze the cell number by using a hemocytometer.</li>
	<li>Seed HEK293 cells in a 6-well plate at a density of 2 x 105 cells per well and incubate for 24 hr.</li>
	<li>For transfection use the protocol provided by the manufacturer of the transfection reagent. Transfect cells with pEGFP-C2 or pEGFP-C2-CaeB using the transfection reagent. Prior to use, warm up the transfection reagent and the media required to RT. Use a 3:1 ratio of transfection reagent to DNA.
	<ol>
		<li>In detail, pipette 1.5 &#181;l of transfection reagent directly into 50 &#181;l of serum-free DMEM medium. For complex formation, pipette 0.5&#160;&#181;g of DNA encoding GFP or GFP-CaeB to the transfection reagent-containing mixture and incubate for 15&#160;min at RT.</li>
		<li>Transfect cells by adding the reaction mixture in a drop-wise manner and incubate at 37&#160;&#176;C in 5%&#160;CO2 for 24 hr.</li>
	</ol>
	</li>
	<li>Add 1&#160;mg/ml geneticin for selection of GFP-positive clones at 37&#160;&#176;C in 5%&#160;CO2.</li>
	<li>After 6&#160;days, isolate single cells by flow cytometry sorting in a 96-well plate harboring medium and HEK293 supernatant in a 1:1 ratio. Generate HEK293 supernatant from cultured confluent HEK293 cells that were centrifuged for 15&#160;min at 4,966 x g.</li>
	<li>On the following day, replace culture medium with medium containing 1.5&#160;mg/ml geneticin. Change media once a week. If the cells form a confluent monolayer, transfer them to a 24-well plate. Sub-cultivate cells twice a week in a ratio of 1:5 in media containing 1.5&#160;mg/ml geneticin. Finally, analyze the percentage of GFP-positive cells by immunoblot analysis and flow cytometry.</li>
</ol>
2. Analysis of Stable Cell Lines by Immunoblot Analysis
<ol>
	<li>Remove the supernatant and wash the adhered cells once with warm PBS. Resuspend the cells in 100 &#181;l of 2x Laemmli sample buffer, heat for 5&#160;min at 95&#160;&#176;C and store at -20&#160;&#176;C.</li>
	<li>Load approximately 4 x 104 cells per sample on a 12% SDS-PAGE gel. Additionally, load 6 &#181;l&#160;of a commercial protein ladder as a molecular weight marker. Run gels in a gel electrophoresis system under a constant voltage of 180&#160;V for 45-60&#160;min with 1x&#160;Laemmli running buffer.</li>
	<li>Blot proteins onto PVDF membranes (pore size of 0.45&#160;&#181;m) at a constant voltage of 16&#160;V for 60&#160;min using a Trans-Blot SD Semi-Dry Transfer Cell.</li>
	<li>Block membranes in 10 ml of 5%&#160;nonfat dry milk in PBS/0.05% Tween 20 (MPT) for 1 hr at RT.</li>
	<li>Dilute 4 &#181;l of anti-GFP antibody in 4 ml of MPT and incubate membranes O/N at 4&#160;&#176;C.</li>
	<li>Perform three 10 min washing steps with 10 ml of PBS/0.05% Tween 20.</li>
	<li>Incubate membranes with 0.8 &#181;l of horseradish peroxidase-conjugated secondary antibodies in 4 ml of MPT.</li>
	<li>After 1 hr incubation at RT, wash membranes three times with PBS/0.05% Tween 20 (as described in 2.6) and detect horseradish peroxidase activity with a commercial kit according to the manufacturer&#8217;s protocol. Apply standard equipment for film development to visualize protein bands.</li>
</ol>
3. Analyze the Cells Using a Flow Cytometer.
<ol>
	<li>Resuspend cells in PBS. Use HEK293 cells as negative control to adjust forward scatter (FSC) and side scatter (SSC) so that the cells are on scale. Draw a gate on living cells by excluding cell doublets, aggregates and cell debris.</li>
	<li>Adjust the photomultiplier tube (PMT) gain so that the unstained cells are on the far left of the histogram for the FL1 channel (488 nm argon laser).</li>
	<li>To analyze the fluorescence intensity of HEK293-GFP and HEK293-GFP-CaeB cells open the histogram of the FL1 channel and acquire 10,000 events on the gated population.</li>
	<li>Analyze the data using commercial FACS analysis software.</li>
</ol>
4. Transfection of the Stable Cell Line with the Inducible Expression Vector System
<ol>
	<li>Seed HEK293 cells stably expressing GFP or GFP-CaeB in a 12-well plate at a density of 1 x 105 cells/well.</li>
	<li>19 hr post-seeding, co-transfect the cells with regulator plasmid and response plasmid either encoding Bax or activated caspase&#160;3.
	<ol>
		<li>Co-transfect the stable HEK293 cells with 100&#160;ng of pWHE125-P regulator plasmid (Figure 2) and 100&#160;ng of the response plasmids pWHE655-hBax or pWHE655-revCasp3 (Figure 3) and 0.4 &#181;l of polyethylenimine.</li>
		<li>Prepare the DNA and polyethylenimine transfection reagent each in 75 &#181;l of Opti-MEM medium and incubate for exactly 5&#160;min at RT. For complex formation, incubate both mixtures together for 15&#160;min at RT.</li>
	</ol>
	</li>
	<li>Add the polyethylenimine/DNA solution drop-wise to the cells and incubate at 37&#160;&#176;C in 5%&#160;CO2.</li>
</ol>
5. Induction of Apoptosis
<ol>
	<li>5 hr post-transfection, induce expression of the pro-apoptotic proteins by addition of 1&#160;&#181;g/ml doxycycline to the culture medium. Incubate cells for 18 hr at 37&#160;&#176;C in 5%&#160;CO2.</li>
</ol>
6. Analysis of Host Cell Apoptosis by Immunoblot Analysis
<ol>
	<li>Inspect cells prior to harvesting under the light microscope to check for cell death induction. Apoptotic cells are detectible by the presence of apoptotic bodies surrounding the dying cell.</li>
	<li>Remove the supernatant and wash the adhered cells once with warm PBS. Resuspend the cells in 100 &#181;l of 2x Laemmli sample buffer, heat for 5&#160;min at 95&#160;&#176;C and store at -20&#160;&#176;C.</li>
	<li>Load approximately 4 x 104 cells per sample on a 12% SDS-PAGE gel. Additionally, load 6 &#181;l of a commercial protein ladder as a molecular weight marker. Run gels in a gel electrophoresis system under a constant voltage of 180&#160;V for 45-60&#160;min with 1x&#160;Laemmli running buffer.</li>
	<li>Blot proteins onto PVDF membranes (pore size of 0.45&#160;&#181;m) at a constant voltage of 16&#160;V for 60&#160;min using a Trans-Blot SD Semi-Dry Transfer Cell.</li>
	<li>Block membranes in 10 ml of 5%&#160;nonfat dry milk in PBS/0.05% Tween 20 (MPT) for 1 hr at RT.</li>
	<li>Dilute 4 &#181;l of anti-cleaved poly ADP-ribose polymerase (PARP) antibody in 4 ml of MPT and incubate O/N at 4&#160;&#176;C.</li>
	<li>Perform three 10 min washing steps with 10 ml of PBS/0.05% Tween 20.</li>
	<li>Incubate membranes with 0.8 &#181;l horseradish peroxidase-conjugated secondary antibodies diluted in 4 ml MPT.</li>
	<li>After 1 hr incubation at RT, wash membranes three times with PBS/0.05% Tween 20 (as described in 6.7) and detect horseradish peroxidase activity with a commercial kit according to manufacturer&#8217;s protocol. Apply standard equipment for film development to visualize protein bands.</li>
	<li>Remove bound antibodies by 30&#160;min incubation at RT with 7 ml Western Blot Stripping Buffer.</li>
	<li>After three washes with PBS/0.05% Tween 20 (as described in 6.7), probe the membranes with 4 &#181;l of anti-actin antibody in 4 ml of MPT O/N at 4&#160;&#176;C.</li>
	<li>After three washing steps with MPT (as described in 6.7), incubate the membranes with 0.8 &#181;l of horseradish peroxidase-conjugated secondary antibodies diluted in 4 ml of MPT.</li>
	<li>After 1 hr incubation at RT, wash membranes three times with PBS/0.05% Tween 20 (as described in 6.7 and detect horseradish peroxidase activity with a commercial kit according to the manufacturer&#8217;s protocol. Apply standard equipment for film development to visualize protein bands. 
	Note: All incubation steps were performed on a plate shaker for the indicated time and temperature.</li>
</ol>

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 disease. Furthermore, as the effector proteins frequently exert their function by mimicking host cell activities, they can also be used to learn more about the biology of eukaryotic cells1. However, studying the biochemical activity of a single effector protein has proven to be complicated for several reasons: 1) functional redundancy among effector proteins, 2) the temporal regulation of engagement of the effector proteins, and 3) the effector protein working in context with another effector protein, interfering with its function. Therefore, the activity of an effector protein is commonly studied by overexpression studies. Unfortunately, the usage of transient overexpression of the effector protein also has several drawbacks. Thus, only single cell analysis assays can be employed, and the activity of the effector protein cannot be analyzed if the activity of the effector protein depends on the activity of another effector protein. To overcome at least the first drawback, stable cell lines can be used. In case the expression of the effector protein is toxic for the cell, this is not suitable. In this scenario, stable and inducible cell lines can be established using a similar system as we describe here for transient expression of apoptosis-inducing proteins (see below). However, our results (Figure 5) demonstrate that the expression of CaeB protects the cells from undergoing apoptosis and is therefore not toxic. There are a few options in dissecting at which step within the apoptotic cascade CaeB interferes with apoptosis induction. One possibility is to determine the host cell binding partner of CaeB. However, this is very challenging and the identification of the binding partner might not help to explain the point of action of CaeB within the apoptotic cascade. Another possibility is to use different apoptosis inducers, but CaeB inhibits apoptosis induction by two different apoptosis inducers, UV-light and staurosporine7, indicating that this approach might also not lead to the identification of the point of action of CaeB. Another possibility in narrowing down the point of action of CaeB, which was exploited here, is to use a system where the apoptosis signaling cascade can be induced at defined steps.
In case expression of the bacterial effector protein that was selected as protein of interest already interferes with cell physiology, its expression can also be placed under control of an inducible expression system, e.g., such as the one used to express the target protein. Then, expression of both protein of interest and target protein will be concomitantly induced when the ligand is added to the cell culture allowing the investigator to monitor the cells for differential effects on cell physiology and signaling system output.
Clearly, expressing a protein of interest and trying to identify its point of action in a specific intracellular pathway by inducible expression of selected pathway proteins works best if the protein of interest interferes with the physiological activity of the selected pathway. In this case, readout is simply the lack of a signal normally associated with the pathway, such as cell death in the case of apoptotic signaling or activation of transcription in the case of Wnt signaling. In principle, proteins that activate a specific pathway can also be probed with this type of assay system. It only has to be set up differently, for example, using inducible expression of a si/sh/miRNA against a pathway protein and testing if the protein of interest can rescue pathway signaling. Alternatively, small molecule inhibitors of specific proteins can be used to interrupt signal transduction within a pathway and then to check for pathway rescue by inducible expression of a protein of interest. However, this type of approach will be experimentally more challenging than the protocol presented above.
The selection of the target gene to be probed in the interference assay is, in principle, straightforward. It is best to test as many of the proteins in the selected signaling pathway as possible to fine tune the identification of the protein of interest&#8217;s site of interaction with the pathway. This should be done in an iterative fashion, because it is not always experimentally feasible or even possible to monitor all potential interaction partners in a single experiment. Therefore, it is best to first test several proteins spaced out evenly along the entire pathway and then to refine the experiment using the information gained in the first round. In signaling pathways, many proteins are modified post-translationally, either by a covalent modification, such as phosphorylation or sumoylation, or by a processing event, such as proteolytic cleavage. Consequently, the respective protein is either in a ground-state or in an activated form. If possible, it is best to test both forms of the protein for two reasons. One is that the activated form delivers a higher output signal31, which presents a more stringent challenge for the protein of interest. In our proof-of-principle example, apoptotic signaling, the caspase 3 is activated by proteolytic cleavage and dimerization. This activation event can be simulated artificially by rearranging the unprocessed pro-caspase gene. The small subunit is artificially placed in front of the large subunit and both are joined by a linker. This is termed a &#8220;reverse caspase&#8221; and represents the activated form of the enzyme when expressed35. It was chosen due to its higher degree of activity. The second reason for probing both ground-state and activated forms of a pathway protein is because it allows one to distinguish if the protein of interest interferes with activation of the target protein, or if it acts downstream of the protein in the pathway. In the first case, cell death will be inhibited if the ground-state form is expressed, but not if the activated form is present. In the second case, neither protein will lead to apoptosis. Thus, testing both forms of a modified protein improves the resolution of the analytic system.
Cloning the target gene in the inducible expression vector requires several decisions. First, shall expression of the target gene occur only transiently or is it better if the vector is stably integrated into the genome of the host cell line? If only transient expression is required, a simple vector such as pUHC13-324, which contains a Tet-dependent promoter and a polyA site, or a bidirectional vector such as pBi-2 or pBi-336, which couples expression of the gene of interest to the expression of a reporter gene for easier monitoring of target gene expression, will be sufficient. If stable expression is preferred, a vector such as pWHE65533 (Figure 3) should be used. It contains insulators that flank the transgene expression unit to prevent position effects at the genomic integration site from interfering with inducible transgene expression. It also contains a linked, but independently expressed, resistance cassette for G418 to facilitate the selection of vector integration events. The net result of these modifications is an increase in the number of clones with excellent regulatory properties33,37,38. cDNA sequences of target genes can be cloned into this vector by using the unique restriction sites for EcoRI and EcoRV33. Cloning of the vector expressing the reverse Caspase-3 cDNA was described in detail in reference 33. Cloning of the vector expressing human Bax was performed analogously. The human bax cDNA was amplified by PCR from the plasmid pVenus-Bax39 using oligonucleotide primers that introduce restriction sites for EcoRI and EcoRV. After purification of the PCR fragment and restriction with the two enzymes, the fragment was ligated with likewise-restricted pWHE655, yielding pWHE655TREtight-Bax. Second, which minimal promoter is most appropriate for transgene expression? The original minimal promoter PhCMV*-124 displays some cell-type specific leakiness30, due in part to the presence of functional interferon-&#945; inducible response elements29. This led to the development of second generation Tet-dependent promoters, termed &#916;MtetO, TREtight and SG-TRE, with altered spacing and sequences between the tetO elements and with different minimal promoter sequences33,40-42. They display greatly reduced leakiness in the absence of doxycycline. Among these second generation promoters, &#916;MtetO has the lowest transgene expression level in the presence of doxycycline33. TREtight mediates an intermediate and SG-TRE the highest level of transgene expression33. Further modifications have minimized background expression of the tetracycline responsive promoter even more43,44. Thus, the choice of a tetracycline-responsive promoter can be made according to the requirements or limitations of the respective transgene.
Independent of the type of experimental approach, tightness of expression of the target protein is important. For example, induction of apoptosis with activated forms of pro-apoptotic proteins leads to cell death, even when only minimal amounts of the respective target protein are expressed31-33. The required stringency is achieved by adding a tetracycline-dependent transsilencer to the conditional regulatory system30. It ensures that transcription from the inducible promoter is actively repressed, which is especially important when transient transfections are performed, since they are typically rather leaky in their transgene expression30. If signaling pathways that do not induce cell death are probed, the requirement for stringent regulation of target gene expression may be relaxed and inducible systems without a transsilencer could be used alternatively43,44.
The inducible regulatory system used to probe the signaling pathway does not have to be the Tet system. Its major advantage is that it has been intensively used in mammalian cells for more than 20 years now and that it is the most highly developed and best characterized inducible control system. Nevertheless, there are at least 25 other conditional regulatory systems45,46 that could be employed alternatively, or in addition to the Tet system. Two or three different inducible systems might be useful in probing convergent signaling pathways or for testing redundancy of either the target gene or the protein of interest.
The procedure presented here should work with transfection efficiencies that result in a robust and reliable output signal from the respective signaling pathway that is probed. Because the stable cell lines express the protein of interest in a homogenous manner (Figure 4B), all cells that are transiently transfected with the inducible plasmid expressing the protein of interest are probed for interference of the protein of interest with pathway signaling.

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><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&amp;nbsp;</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 were also analyzed by flow cytometry. As shown in Figure 4B, over 90% of the cells in both cell lines expressed GFP. Thus, these stable cell lines can be used to further analyze the function of CaeB. In the next step, the anti-apoptotic activity of CaeB was assayed by immunoblot analysis using an antibody against cleaved PARP. Proteolytic cleavage of nuclear PARP inactivates DNA repair activity and is a typical marker of the terminal stages of apoptosis. Cleavage of PARP is not detected in HEK293-GFP or HEK293-GFP-CaeB cells, demonstrating that neither the expression of GFP, nor of GFP-CaeB is toxic to the cells. However, when the cells were treated with staurosporine, a potent inducer of intrinsic apoptosis, cleavage of PARP was detected (Figure 5). Importantly, HEK293-GFP-CaeB cells exhibit reduced cleavage of PARP, indicating the anti-apoptotic activity of the Coxiella burnetii type IV secretion system effector protein CaeB7.
To analyze at which step CaeB interferes with the apoptotic pathway, the Tet-On system was employed. In detail, HEK293-GFP or HEK293-GFP-CaeB cells were transfected with a regulator plasmid constitutively expressing a doxycycline-controlled dual repressor/activator setup (Figure 2) and a pTRE response plasmid encoding either full length human Bax or the activated form of human caspase 3, termed revCasp 3 (Figure 3). This system is tightly regulated, as demonstrated by the lack of PARP cleavage under non-inducing conditions (Figure 6). Addition of doxycycline to the transfected cells resulted in the expression of Bax or revCasp 3. If CaeB interferes with the apoptotic pathway downstream of Bax, then the apoptotic signal generated by expression and subsequent activation of Bax should be blocked by CaeB expression. Indeed, HEK293 cells stably expressing GFP-CaeB profoundly inhibit apoptosis induction by doxycycline-controlled Bax expression, while HEK293-GFP cells displayed obvious PARP cleavage. This result indicates that CaeB blocks apoptosis induction downstream of Bax activation. Next, it was analyzed whether CaeB can interfere with caspase 3 activation &#8212; a late event in the apoptotic pathway. HEK293-GFP cells, as well as HEK293-GFP-CaeB cells, exhibit PARP cleavage (Figure 6), indicating that once the effector caspase 3 is activated, CaeB cannot prevent apoptosis from occurring. Taken together, these data demonstrate that CaeB acts between Bax and caspase 3 activation.
<img alt="Figure 1" src="/files/ftp_upload/52903/52903fig1.jpg" /> 
Figure 1.&#160;Schematic overview of the tetracycline regulatory system. The Tet-On system is composed of two different plasmids, the regulatory and response plasmids. The regulatory plasmid encodes the Tet-responsive transsilencer (tTS; filled circle) and the Tet-responsive reverse transactivator (rtTA, open circle). Both proteins are constitutively expressed under the control of the strong, constitutive elongation factor-1 alpha promoter (PEF-1a). TTS and rtTA are chimeric transcription factors consisting of a eukaryotic transcriptional regulatory domain (silencer or activator, respectively) and a bacterial tetracycline repressor (TetR). TetR mediates DNA binding to seven tet operator (tetO) sequences on the response plasmid. TetO is located upstream of the inducible minimal cytomegalovirus promoter (PCMV), together forming the Tet-responsive element (TRE). Under non-inducing conditions, tTS is bound to TRE preventing expression. After addition of doxycycline (Dox; filled diamond), rtTA interacts with Dox leading to conformational changes and activation. Activated rtTA replaces tTS on the response plasmid, allowing transcription of the respective target genes Bax and caspase&#160;3, thus leading to apoptosis induction and cell death. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52903/52903fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52903/52903fig2.jpg" /> 
Figure 2.&#160;Schematic overview of the regulatory plasmid pWHE125. Elements essential for propagation in bacteria, including an origin of replication (ori pBR) and an antibiotic resistance cassette against ampicillin (AmpR), and for expression of the Tet-transregulators, such as the strong, constitutive immediate early promoter/enhancer of the human Cytomegalovirus (P/EhCMV), an internal ribosome binding site from polio-virus (PV-IRES) and a polyA-site from Simian virus 40 (SV40 polyA) are displayed. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52903/52903fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52903/52903fig3.jpg" /> 
Figure 3.&#160;Schematic overview of the response plasmid. Elements essential for propagation in bacteria, including an origin of replication (ori pBR) and an antibiotic resistance cassette (AmpR), and for inducible expression in eukaryotes, such as the transgene expression cassette with the Tet-dependent minimal promoter TREtight (PTREtight), the gene of interest human Bax (hBax) and a polyA site from Simian virus 40 (SV40 polyA), are depicted. The transgene expression cassette is flanked by tandem direct repeats of two copies of the chicken HS4 insulator core sequence (HS4 core). Additionally, a G418 resistance cassette consisting of a murine phosphoglycerate kinase 1 promoter (PmPGK), a neomycin resistance gene (G418R) and a human thymidine kinase polyA site (TK polyA) is present. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52903/52903fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/52903/52903fig4.jpg" /> 
Figure 4.&#160;HEK293-GFP and HEK293-GFP-CaeB stably express green fluorescent proteins. (A) Whole-cell lysates of HEK293-GFP and HEK293-GFP-CaeB cells were immunoblotted for GFP to show stable expression. (B) Representative flow cytometry (FACS) analysis of HEK293-GFP and HEK293-GFP-CaeB cells is shown to demonstrate the intensity of the GFP expression. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52903/52903fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/52903/52903fig5.jpg" /> 
Figure 5.&#160;Effects of the expression of GFP-CaeB on staurosporine-induced apoptosis. HEK293-GFP and HEK293-GFP-CaeB cells were treated with 4 &#181;M staurosporine for 6 hr to induce intrinsic apoptosis. Apoptosis was analyzed by cleaved PARP immunoblot analysis. PARP cleavage was reduced in HEK293-GFP-CaeB cells in comparison to HEK293-GFP cells. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52903/52903fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/52903/52903fig6.jpg" /> 
Figure 6.&#160;Usage of the TetOn system to narrow down the point of action of CaeB. (A) Apoptosis was induced by expression of Bax in HEK293-GFP and HEK293-GFP-CaeB cells. Apoptosis was analyzed by cleaved PARP immunoblot analysis. PARP cleavage was reduced in HEK293-GFP-CaeB cells in comparison to HEK293-GFP cells. (B) Apoptosis was induced by expression of revCasp 3 in HEK293-GFP and HEK293-GFP-CaeB cells. Apoptosis was analyzed by cleaved PARP immunoblot analysis. PARP cleavage was comparable in HEK293-GFP-CaeB and HEK293-GFP cells. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52903/52903fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

9.1K Views.  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.

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

A Fluorescence-based Method to Study Bacterial Gene Regulation in Infected Tissues

Bacterial virulence genes are often regulated at the transcriptional level by multiple factors that respond to different environmental signals. Some factors act directly on virulence genes; others control pathogenesis by adjusting the expression of downstream regulators or the accumulation of signals that affect regulator activity. While regulation has been studied extensively during in vitro growth, relatively little is known about how gene expression is adjusted during infection. Such information is important when a particular gene product is a candidate for therapeutic intervention. Transcriptional approaches like quantitative, real-time RT-PCR and RNA-Seq are powerful ways to examine gene expression on a global level but suffer from many technical challenges including low abundance of bacterial RNA compared to host RNA, and sample degradation by RNases. Evaluating regulation using fluorescent reporters is relatively easy and can be multiplexed with fluorescent proteins with unique spectral properties. The method allows for single-cell, spatiotemporal analysis of gene expression in tissues that exhibit complex three-dimensional architecture and physiochemical gradients that affect bacterial regulatory networks. Such information is lost when data are averaged over the bulk population. Herein, we describe a method for quantifying gene expression in bacterial pathogens in situ. The method is based on simple tissue processing and direct observation of fluorescence from reporter proteins. We demonstrate the utility of this system by examining the expression of Staphylococcus aureus thermonuclease (nuc), whose gene product is required for immune evasion and full virulence ex vivo and in vivo. We show that nuc-gfp is strongly expressed in renal abscesses and reveal heterogeneous gene expression due in part to apparent spatial regulation of nuc promoter activity in abscesses fully engaged with the immune response. The method can be applied to any bacterium with a manipulatable genetic system and any infection model, providing valuable information for preclinical studies and drug development.

Described here is a method for analyzing bacterial gene expression in animal tissues at a cellular level. This method provides a resource for studying the phenotypic diversity occurring within a bacterial population in response to the tissue environment during an infection.

Bacterial virulence genes are often regulated at the transcriptional level by multiple factors that respond to different environmental signals. Some ...

Genetics

Tractable Mammalian Cell Infections with Protozoan-primed Bacteria

Many intracellular bacterial pathogens use freshwater protozoans as a natural reservoir for proliferation in the environment. Legionella pneumophila, the causative agent of Legionnaires' pneumonia, gains a pathogenic advantage over in vitro cultured bacteria when first harvested from protozoan cells prior to infection of mammalian macrophages. This suggests that important virulence factors may not be properly expressed in vitro. We have developed a tractable system for priming L. pneumophila through its natural protozoan host Acanthamoeba castellanii prior to mammalian cell infection. The contribution of any virulence factor can be examined by comparing intracellular growth of a mutant strain to wild-type bacteria after protozoan priming. GFP-expressing wild-type and mutant L. pneumophila strains are used to infect protozoan monolayers in a priming step and allowed to reach late stages of intracellular growth. Fluorescent bacteria are then harvested from these infected cells and normalized by spectrophotometry to generate comparable numbers of bacteria for a subsequent infection into mammalian macrophages. For quantification, live bacteria are monitored after infection using fluorescence microscopy, flow cytometry, and by colony plating. This technique highlights and relies on the contribution of host cell-dependent gene expression by mimicking the environment that would be encountered in a natural acquisition route. This approach can be modified to accommodate any bacterium that uses an intermediary host as a means for gaining a pathogenic advantage.

This technique provides a method to harvest, normalize and quantify intracellular growth of bacterial pathogens that are pre-cultivated in natural protozoan host cells prior to infections of mammalian cells. This method can be modified to accommodate a wide variety of host cells for the priming stage as well as target cell types.

Many intracellular bacterial pathogens use freshwater protozoans as a natural reservoir for proliferation in the environment. Legionella pneumophila, the ...

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Bacterial intracellular pathogens can be conceived as molecular tools to dissect cellular signaling cascades due to their capacity to exquisitely manipulate and subvert cell functions which are required for the infection of host target tissues. Among these bacterial pathogens, Listeria monocytogenes is a Gram positive microorganism that has been used as a paradigm for intracellular parasitism in the characterization of cellular immune responses, and which has played instrumental roles in the discovery of molecular pathways controlling cytoskeletal and membrane trafficking dynamics. In this article, we describe a robust microscopical assay for the detection of late cellular infection stages of L. monocytogenes based on the fluorescent labeling of InlC, a secreted bacterial protein which accumulates in the cytoplasm of infected cells; this assay can be coupled to automated high-throughput small interfering RNA screens in order to characterize cellular signaling pathways involved in the up- or down-regulation of infection.

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Listeria monocytogenes is a Gram positive bacterial pathogen frequently used as a major model for the study of intracellular parasitism. Imaging late L. monocytogenes infection stages within the context of small-interfering RNA screens allows for the global study of cellular pathways required for bacterial infection of target host cells.

Bacterial intracellular  pathogens can be conceived as molecular tools to dissect cellular signaling  cascades due to their capacity to exquisitely ...

Dissecting Innate Immune Signaling in Viral Evasion of Cytokine Production

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. Conversely, viruses have evolved intricate strategies to evade and exploit host immune signaling for survival and propagation. Viral immune evasion, entailing host defense and viral evasion, provides one of the most fascinating and dynamic interfaces to discern the host-virus interaction. These studies advance our understanding in innate immune regulation and pave our way to develop novel antiviral therapies.
Murine &#947;HV68 is a natural pathogen of murine rodents. &#947;HV68 infection of mice provides a tractable small animal model to examine the antiviral response to human KSHV and EBV of which perturbation of in vivo virus-host interactions is not applicable. Here we describe a protocol to determine the antiviral cytokine production. This protocol can be adapted to other viruses and signaling pathways.
Recently, we have discovered that &#947;HV68 hijacks MAVS and IKK&#946;, key innate immune signaling components downstream of the cytosolic RIG-I and MDA5, to abrogate NF&#922;B activation and antiviral cytokine production. Specifically, &#947;HV68 infection activates IKK&#946; and that activated IKK&#946; phosphorylates RelA to accelerate RelA degradation. As such, &#947;HV68 efficiently uncouples NF&#922;B activation from its upstream activated IKK&#946;, negating antiviral cytokine gene expression. This study elucidates an intricate strategy whereby the upstream innate immune activation is intercepted by a viral pathogen to nullify the immediate downstream transcriptional activation and evade antiviral cytokine production.

We describe a protocol to measure the antiviral cytokine production in mice infected with a model herpesvirus, murine gamma herpesvirus 68 (&#947;HV68) that is closely-related to human Kaposi&#8217;s sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). Utilizing genetically modified mouse strains and mouse embryonic fibroblasts (MEFs), we assessed the antiviral cytokine production both in vivo and ex vivo. &#8220;Reconstituting&#8221; the expression of innate immune components in knockout embryonic fibroblasts by lentiviral transduction, we further pinpoint specific innate immune molecules and dissect the key signaling events that differentially regulate the antiviral cytokine production.

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. ...

Monitoring the Assembly of a Secreted Bacterial Virulence Factor Using Site-specific Crosslinking

This article describes a method to detect and analyze dynamic interactions between a protein of interest and other factors in vivo. Our method is based on the amber suppression technology that was originally developed by Peter Schultz and colleagues1. An amber mutation is first introduced at a specific codon of the gene encoding the protein of interest. The amber mutant is then expressed in E. coli together with genes encoding an amber suppressor tRNA and an amino acyl-tRNA synthetase derived from Methanococcus jannaschii. Using this system, the photo activatable amino acid analog p-benzoylphenylalanine (Bpa) is incorporated at the amber codon. Cells are then irradiated with ultraviolet light to covalently link the Bpa residue to proteins that are located within 3-8 &#197;. Photocrosslinking is performed in combination with pulse-chase labeling and immunoprecipitation of the protein of interest in order to monitor changes in protein-protein interactions that occur over a time scale of seconds to minutes. We optimized the procedure to study the assembly of a bacterial virulence factor that consists of two independent domains, a domain that is integrated into the outer membrane and a domain that is translocated into the extracellular space, but the method can be used to study many different assembly processes and biological pathways in both prokaryotic and eukaryotic cells. In principle interacting factors and even specific residues of interacting factors that bind to a protein of interest can be identified by mass spectrometry.

This article illustrates the use of pulse-chase radio labeling in combination with site-specific photocrosslinking to monitor interactions between a protein of interest and other factors in E. coli. Unlike traditional chemical cross-linking methods, this approach generates high resolution &#8220;snapshots&#8221; of an ordered assembly pathway in a living cell.

This article describes a method to detect and analyze dynamic interactions between a protein of interest and other factors in vivo. Our method is based on ...

Applying Fluorescence Resonance Energy Transfer (FRET) to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a replicative niche. A cohort of effectors are translocated through this system into the host cell to manipulate host processes and allow the establishment of a unique lysosome-derived vacuole for replication. The method presented here involves the combination of two well-established techniques: specific gene silencing using siRNA and measurement of effector translocation using a FRET-based substrate that relies on &#946;-lactamase activity. Applying these two approaches, we can begin to understand the role of host factors in bacterial secretion system function and effector translocation. In this study we examined the role of Rab5A and Rab7A, both important regulators of the endocytic trafficking pathway. We demonstrate that silencing the expression of either protein results in a decrease in effector translocation efficiency. These methods can be easily modified to examine other intracellular and extracellular pathogens that also utilize secretion systems. In this way, a global picture of host factors involved in bacterial effector translocation may be revealed.

Applying Fluorescence Resonance Energy Transfer FRET to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Investigating the interactions between bacterial pathogens and their hosts is an important area of biological research. Here, we describe the necessary techniques to measure effector translocation by Coxiella burnetii during siRNA gene silencing using BlaM substrate.

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a ...

Designing Tetracycline-Controlled Synthetic Circuits for Leishmaniasis Therapy

Immunometabolic Circuits in Infection for Advancing Host Directed Therapies

Immuno-metabolism is a pivotal determinant in the progression of leishmaniasis. Synthetic biology-based approach has garnered significant attention as a step toward therapeutic intervention targeting host-associated factors that drive leishmaniasis. Synthetic biology entails the engineering of genetic components in an orthogonal and modular manner to precisely modulate biological systems, imparting novel functions to cells. In the presented study, elucidation of a systematic pipeline for the development of an inducible tetracycline-controlled (TetON)-based synthetic circuit was aimed at delivering succinate dehydrogenase as a therapeutic agent to facilitate the elimination of intracellular Leishmania parasites. The outlined protocol describes the designing of a synthetic circuit and its subsequent validation. The proposed strategy also concentrates on the incorporation of synthetic circuits in the plasmid backbone as a delivery vehicle. Additionally, delivery machinery employing polyplexes-based nano-particles for the delivery of synthetic circuits was used in murine macrophage cell lines without compromising the cellular morphology. Standardization of the method was conducted for selecting transfected cells and determining optimal induction concentration for synthetic circuit expression. Observations reveal a distinct reduction in intracellular parasite burden in transfected cells compared to infected cells. Pro-inflammatory cytokines were expressed post-infection in synthetic circuit transfected and induced cells as a mechanism to promote parasite elimination. This underscores the synthetic biology-based method as a potent approach in leishmaniasis by targeting host factors associated with disease progression.

Here, a protocol for designing, constructing, and delivering tetracycline-controlled based inducible synthetic circuit is presented. The effect of the synthetic circuits on parasite elimination and cytokine expression can be studied by channelizing the presented pipeline. This protocol is relevant for researchers studying synthetic circuit-based therapeutics in infectious diseases.

Immuno-metabolism is a pivotal determinant in the progression of leishmaniasis. Synthetic biology-based approach has garnered significant attention as a ...

Bioengineering

A Hydroponic Co-cultivation System for Simultaneous and Systematic Analysis of Plant/Microbe Molecular Interactions and Signaling

An experimental design mimicking natural plant-microbe interactions is very important to delineate the complex plant-microbe signaling processes. Arabidopsis thaliana-Agrobacterium tumefaciens provides an excellent model system to study bacterial pathogenesis and plant interactions. Previous studies of plant-Agrobacterium interactions have largely relied on plant cell suspension cultures, the artificial wounding of plants, or the artificial induction of microbial virulence factors or plant defenses by synthetic chemicals. However, these methods are distinct from the natural signaling in planta, where plants and microbes recognize and respond in spatial and temporal manners. This work presents a hydroponic cocultivation system where intact plants are supported by metal mesh screens and cocultivated with Agrobacterium. In this cocultivation system, no synthetic phytohormone or chemical that induces microbial virulence or plant defense is supplemented. The hydroponic cocultivation system closely resembles natural plant-microbe interactions and signaling homeostasis in planta. Plant roots can be separated from the medium containing Agrobacterium, and the signaling and responses of both the plant hosts and the interacting microbes can be investigated simultaneously and systematically. At any given timepoint/interval, plant tissues or bacteria can be harvested separately for various &#34;omics&#34; analyses, demonstrating the power and efficacy of this system. The hydroponic cocultivation system can be easily adapted to study: 1) the reciprocal signaling of diverse plant-microbe systems, 2) signaling between a plant host and multiple microbial species (i.e.&#160;microbial consortia or microbiomes), 3) how nutrients and chemicals are implicated in plant-microbe signaling, and 4) how microbes interact with plant hosts and contribute to plant tolerance to biotic or abiotic stresses.

The described hydroponic cocultivation system supports intact plants with metal mesh screens and cocultivates them with bacteria. Plant tissue, bacteria, and secreted molecules can then be separately harvested for downstream analyses, simultaneously allowing for the molecular responses of both plant hosts and interacting microbes or microbiomes to be investigated.

An experimental design mimicking natural plant-microbe interactions is very important to delineate the complex plant-microbe signaling processes. ...

Biology

Live Cell Fluorescence Microscopy to Observe Essential Processes During Microbial Cell Growth

Core cellular processes such as DNA replication and segregation, protein synthesis, cell wall biosynthesis, and cell division rely on the function of proteins which are essential for bacterial survival. A series of target-specific dyes can be used as probes to better understand these processes. Staining with lipophilic dyes enables the observation of membrane structure, visualization of lipid microdomains, and detection of membrane blebs. Use of fluorescent-d-amino acids (FDAAs) to probe the sites of peptidoglycan biosynthesis can indicate potential defects in cell wall biogenesis or cell growth patterning. Finally, nucleic acid stains can indicate possible defects in DNA replication or chromosome segregation. Cyanine DNA stains label living cells and are suitable for time-lapse microscopy enabling real-time observations of nucleoid morphology during cell growth. Protocols for cell labeling can be applied to protein depletion mutants to identify defects in membrane structure, cell wall biogenesis, or chromosome segregation. Furthermore, time-lapse microscopy can be used to monitor morphological changes as an essential protein is removed and can provide additional insights into protein function. For example, the depletion of essential cell division proteins results in filamentation or branching, whereas the depletion of cell growth proteins may cause cells to become shorter or rounder. Here, protocols for cell growth, target-specific labeling, and time-lapse microscopy are provided for the bacterial plant pathogen Agrobacterium tumefaciens. Together, target-specific dyes and time-lapse microscopy enable characterization of essential processes in A. tumefaciens. Finally, the protocols provided can be readily modified to probe essential processes in other bacteria.

Understanding the function of essential processes in bacteria is challenging. Fluorescence microscopy with target-specific dyes can provide key insights into microbial cell growth and cell cycle progression. Here, Agrobacterium tumefaciens is used as a model bacterium to highlight methods for live cell imaging for characterization of essential processes.

Core cellular processes such as DNA replication and segregation, protein synthesis, cell wall biosynthesis, and cell division rely on the function of ...

Split Green Fluorescent Protein System to Visualize Effectors Delivered from Bacteria During Infection

Bacteria, one of the most important causative agents of various plant diseases, secrete a set of effector proteins into the host plant cell to subvert the plant immune system. During infection cytoplasmic effectors are delivered to the host cytosol via a type III secretion system (T3SS). After delivery into the plant cell, the effector(s) targets the specific compartment(s) to modulate host cell processes for survival and replication of the pathogen. Although there has been some research on the subcellular localization of effector proteins in the host cells to understand their function in pathogenicity by using fluorescent proteins, investigation of the dynamics of effectors directly injected from bacteria has been challenging due to the incompatibility between the T3SS and fluorescent proteins.
Here, we describe our recent method of an optimized split superfolder green fluorescent protein system (sfGFPOPT) to visualize the localization of effectors delivered via the bacterial T3SS in the host cell. The sfGFP11 (11th &#946;-strand of sfGFP)-tagged effector secreted through the T3SS can be assembled with a specific organelle targeted sfGFP1-10OPT (1-10th &#946;-strand of sfGFP) leading to fluorescence emission at the site. This protocol provides a procedure to visualize the reconstituted sfGFP fluorescence signal with an effector protein from Pseudomonas syringae in a particular organelle in the Arabidopsis and Nicotiana benthamiana plants.

Fluorescent protein-based approaches to monitor effectors secreted by bacteria into host cells are challenging. This is due to the incompatibility between fluorescent proteins and the type-III secretion system. Here, an optimized split superfolder GFP system is used for visualization of effectors secreted by bacteria into the host plant cell.

Bacteria, one of the most important causative agents of various plant diseases, secrete a set of effector proteins into the host plant cell to subvert the ...

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

Summary

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Chapters in this video

A Fluorescence-based Method to Study Bacterial Gene Regulation in Infected Tissues

Tractable Mammalian Cell Infections with Protozoan-primed Bacteria

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Dissecting Innate Immune Signaling in Viral Evasion of Cytokine Production

Monitoring the Assembly of a Secreted Bacterial Virulence Factor Using Site-specific Crosslinking

Applying Fluorescence Resonance Energy Transfer (FRET) to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Immunometabolic Circuits in Infection for Advancing Host Directed Therapies

A Hydroponic Co-cultivation System for Simultaneous and Systematic Analysis of Plant/Microbe Molecular Interactions and Signaling

Live Cell Fluorescence Microscopy to Observe Essential Processes During Microbial Cell Growth

Split Green Fluorescent Protein System to Visualize Effectors Delivered from Bacteria During Infection