We thank Dr. Antonio Virgilio&#160;Failla for providing the FRET acquisition quantification algorithm and Erwin Bohn for providing the pMK-bla and pMK-ova constructs.

1. Peak Emission and Cross-talk Determination (See Also Figure&#160;2)
<ol>
	<li>In order to determine the emission peaks and amount of cross-talk between the donor (coumarin derivate V450) and acceptor (fluorescein) dyes, perform spectral scans of cells labeled with both individual fluorophores at 405 nm excitation.</li>
	<li>Determine a 10 nm bandwidth within the peak of each dye that will be used for the donor and acceptor channels (here 455-465 nm and 525-535 nm). Plot the normalized intensity over the wavelength and calculate the average percentage of cross-talk of the donor dye in the acceptor channel and vice versa (Figure&#160;2).</li>
</ol>
2. Preparation of Y. enterocolitica Strains for Cell Culture Infection
<ol>
	<li>Use the following strains: WA-314 pMK-ova (WA-314 transformed with the plasmids pMK-bla17 and pMK-ova17, respectively) and WA-314&#916;YopE pMK-bla (WA-314&#916;YopE transformed with the plasmid pMK-bla17)</li>
	<li>At least two days before infection streak Y. enterocolitica strains WA-314 pMK-ova, WA-314 pMK-bla and WA-314&#916;E pMK-bla from cryo stocks on LB-agar plates containing the required antibiotics (kanamycin for WA-314 pMK-bla and WA-314 pMK-ova; kanamycin and tetracycline for WA-314&#916;E pMK-bla) and grow O/N at 27 &#176;C. Afterwards keep the plates for up to one week at 4 &#176;C.</li>
	<li>One day before infection inoculate 3 ml of LB-medium containing the required antibiotics with one colony of the respective strains and incubate O/N at 27 &#176;C in a shaker at 180 rpm.</li>
	<li>On the day of infection inoculate 2 ml of the O/N culture into 40 ml of fresh LB-medium without antibiotics and incubate for 90 min at 37 &#176;C in a shaker at 180 rpm to induce expression of the type III secretion system.</li>
	<li>Transfer the cultures into a suitable tube and keep the cultures on ice or at 4 &#176;C from now on. Centrifuge the cultures for 10 min at 5,000 x g and 4 &#176;C.</li>
	<li>Resuspend the bacterial pellet in 2-3 ml of ice cold PBS. Dilute the bacterial suspension to a concentration of 8 x 108 cfu/ml by using a spectrophotometer. Determine the corresponding optical density individually. Keep this suspension on ice until infecting the HeLa cells.</li>
</ol>
3. Plating HeLa Cells
<ol>
	<li>One day before infection seed 1.5 x 104 cells HeLa cells in 200 &#181;l DMEM containing 10% heat inactivated fetal calf serum (FCS) in wells of a 96 well plate and cultivate in a 5% CO2 incubator at 37 &#176;C. Seed three wells for each strain (three different incubation times).</li>
</ol>
4. Infection of HeLa Cells and Loading with CCF4
<ol>
	<li>Infect HeLa cells by adding 3.5 &#181;l of the bacterial suspension to the medium and mix by gently pipetting the medium twice. Allow the bacteria to sediment onto the cells at a MOI (moiety of infection) of about 100 bacteria per cell. For a time course start infections at -90 min, -60 min and -30 min and incubate at 37 &#176;C in a 5% CO2 incubator until 0 min to achieve a common end point.</li>
	<li>When incubation is completed, carefully aspirate the medium without removing HeLa cells and add 50 &#181;l of PBS containing 20 &#181;g/ml chloramphenicol to stop expression of effectors and 2.5 mM probenecid to reduce export of CCF4.</li>
	<li>At this point transfer the 96-well plate into the microscope stage and define suitable spots for later analysis in each well. Finish this step within 10 min (for details of acquisition see section 5.1).</li>
	<li>Per well, add 70 &#181;l of CCF4/AM loading solution (0.24 &#181;l solution A, 1.2 &#181;l solution B, 18.56 &#181;l solution C (solution A, B, and C provided within CCF4/AM loading kit) and 50 &#181;l PBS (containing 20 &#181;g/ml chloramphenicol and 2.5 mM probenecid) using a multi pipette and incubate for 5 min at RT. From now on work without interruptions.</li>
	<li>Aspirate the loading solution and add 100 &#181;l of PBS containing 20 &#181;g/ml chloramphenicol and 2.5 mM probenecid using a multi pipette. Then rapidly transfer the 96 well plate into the microscope stage and start acquisition within 10 min from aspiration of the loading solution.</li>
</ol>
5. Laser Scanning Microscopy and Analysis of Data
<ol>
	<li>Image acquisition
	<ol>
		<li>Set the imaging conditions in a way to maximize the total photon count and minimize phototoxicity, photobleaching and cross-excitation.
		<ol>
			<li>In a modern confocal laser scanning microscope, use a 20X high numerical aperture immersion objective, open the detector pinhole to 2.5 Airy units (i.e., wide optical sectioning), choose high sensitivity GaAsP-detectors with simultaneously active donor and acceptor channels, 8 bit depth, ~750 nm pixel size, 3-fold frame averaging and excite with 10% of a 405 nm diode laser.</li>
			<li>To ensure correct quantification set the detector gain of both channels to the same value and to avoid any saturated pixels. 
			Note: Here, the gains were set to 150%.</li>
		</ol>
		</li>
		<li>Ensure continuous immersion by spreading immersion medium to the bottom of the wells, for example with a 1 ml syringe, and avoid trapping of any air bubbles.</li>
		<li>Activate the transmitted light and find a representative field of view in every well. Use a correctly calibrated automatic stage and mark the position in the software.</li>
		<li>After removal of the plate for staining (see section 4.3), reinsert the plate and add a weight on top to avoid focal drift. Review the saved positions with the laser scanning mode and adjust the focus and lateral position properly.</li>
		<li>Set the time lapse to 2 min, the duration to 2 hr and start acquisition.</li>
	</ol>
	</li>
	<li>Image quantification (examples will be given for Bitplane Software such as Imaris)
	<ol>
		<li>Import the dataset into software that allows arithmetic calculations of pixel matrices. Do this by dragging and dropping the source file containing both the donor and the acceptor channel on the software icon.</li>
		<li>Subtract the background in both channels using the same offset. To do this, click Menu and then click Image&#62;Processing&#62;Baseline Subtraction. Select each channel and enter the baseline value.</li>
		<li>Correct the bleed-through of the donor channel (Ch1) into the acceptor channel (Ch2) with the pre-determined correction factor f (see 1.1), creating a new channel: 
		Ch2&#8217; = Ch2 &#8211; Ch1 * f 
		NOTE: A bleed-through of the acceptor into the donor channel was not detectable over the level of noise.
		<ol>
			<li>Click Menu&#62;Image Processing&#62;Channel Arithmetics and enter abs(ch2-(ch1*0.33)). Afterwards delete Channel 2 by clicking Menu&#62;Edit&#62;Delete Channels. Ensure that the bleed-through corrected acceptor channel remains as the new Channel 2. Optional: Change the channel color from gray to the original color by going to Menu&#62;Edit&#62;Image Properties. Select Channel 4&#62;Mapped Color&#62;Base Color and change it to e.g., green.</li>
		</ol>
		</li>
		<li>Create a new channel with the following operation to determine the relative FRET coefficient: 
		Ch3 = Ch2&#8217; / (Ch1 + Ch2&#8217;)
		<ol>
			<li>Go to Menu&#62;Image Processing&#62;Channel Arithmetics and enter ch2./(ch1+ch2)</li>
		</ol>
		</li>
		<li>For proper visualization of the FRET channel in an 8 bit image, create a fourth channel: 
		Ch4 = 255 * Ch3
		<ol>
			<li>Go to Menu&#62;Image Processing&#62;Channel Arithmetics and enter 255*ch3. Change the channel color by going to Menu&#62;Edit&#62;Image Properties. Select Channel 4&#62;Mapped Color&#62;Color Table File&#62;Jet.</li>
		</ol>
		</li>
		<li>Apply a 3x3 median filter to channels Ch3 and Ch4 that will reduce the noise in the images. To do this, click to Menu&#62;Image Processing&#62;Smoothing&#62;Median Filter. Select both channels and apply a filter size &#8220;3x3x1&#8221;.</li>
		<li>For quantification of cellular signals, detect the cells in Ch4 by properly setting the offset and filter the structures by size to exclude too small structures.
		<ol>
			<li>Select the Surpass View and click on the icon &#8220;Add new Surfaces&#8221;. Define the detection algorithm parameters in the Surface window. For faster processing, activate the two checkboxes &#8220;Segment only a Region of Interest&#8221; and &#8220;Process entire Image finally&#8221;. Define the region of interest by editing its dimensions.</li>
			<li>Select Channel 4 as the source channel and set the threshold by Thresholding&#62;Background Subtraction (Local Contrast) and set diameter to 10 &#181;m. Set the minimum intensity threshold manually to 0 and the maximum threshold to the maximum intensity. Filter the structures by area and set the minimum value to e.g., 187 &#181;m&#178;. Finish the surface detection.</li>
		</ol>
		</li>
		<li>Extract the mean values for donor fluorescence intensity (Ch1) and relative FRET coefficient (Ch3) and their standard deviations in the cellular entity. To do this, go to the Surface properties. Change the appearance of the structures by selecting Surfaces Style/Quality&#62;Surface. Select all structures by clicking on the filters tab. Unify the detected structures by going to Process Selection&#62;Unify. Export the surface statistics to MS Excel by clicking in the statistics tab&#62;Export All Statistics to File.</li>
		<li>Plot these values over time. Identify the 10 min interval of maximum slope of donor channel fluorescence increase for each condition as a reasonable parameter to represent the amount of translocated effector. Calculate the slope as m =&#160; &#916; donor fluorescence intensity / &#916; time (min).</li>
	</ol>
	</li>
</ol>

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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+imaging+of+type+III+secretion:+Salmonella+SipA+injection+into+host+cells.">Real-time imaging of type III secretion: Salmonella SipA injection into host cells.</a> Proc Natl Acad Sci U S A. 102 (35), 12548-12553 (2005).</li><li>Charpentier, X., Oswald, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+the+secretion+and+translocation+domain+of+the+enteropathogenic+and+enterohemorrhagic+Escherichia+coli+effector+Cif,+using+TEM-1+beta-lactamase+as+a+new+fluorescence-based+reporter.">Identification of the secretion and translocation domain of the enteropathogenic and enterohemorrhagic Escherichia coli effector Cif, using TEM-1 beta-lactamase as a new fluorescence-based reporter.</a> J Bacteriol. 186 (16), 5486-5495 (2004).</li><li>Koberle, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yersinia+enterocolitica+targets+cells+of+the+innate+and+adaptive+immune+system+by+injection+of+Yops+in+a+mouse+infection+model.">Yersinia enterocolitica targets cells of the innate and adaptive immune system by injection of Yops in a mouse infection model.</a> PLoS Pathog. 5 (8), e1000551 (2009).</li><li>Geddes, K., Cruz, F., Heffron, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+cells+targeted+by+Salmonella+type+III+secretion+in+vivo.">Analysis of cells targeted by Salmonella type III secretion in vivo.</a> PLoS Pathog. 3 (12), e196 (2007).</li><li>Marketon, M. 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The authors declare that they have no competing financial interests.

We here successfully applied a TEM-1 beta-lactamase reporter based assay for quantitative analysis of effector translocation by Y. enterocolitica. Many different variations of this sensitive, specific and relatively straightforward technique have been described in the literature. In this study laser scanning microscopy was conducted for most sensitive and precise detection of fluorescence signals. Specifically the correction for cross-talk between the donor and acceptor dyes and the individually adjustable detection bandwidths allow for superior accuracy of measurement compared to other variations of the method. The results clearly show that the method is capable of quantitatively analyzing translocation. Different incubation times representing different concentrations of intracellular beta-lactamase were positively correlated with the slope of donor fluorescence intensity. Also a significantly higher translocation rate of the WA-314&#916;YopE compared to the wild type strain WA-314 could be demonstrated.
Two alternative options for data analysis were applied: Donor channel intensities and relative FRET coefficients. The advantage of using the donor channel is that it directly represents the accumulation of a CCF-4 hydrolysis product (coumarin). This approach is possible in a microscopic setup, because other than in a plate reader correction for differences in cell densities by using a ratio of donor/acceptor is not necessary. However, export of the CCF4 dye or its hydrolysis products is still a possible confounder in this approach. This disadvantage can be overcome by calculating the relative FRET coefficient.
In the described setup experiments were conducted in 96 well plates. This format allows for testing of multiple conditions in a single experiment and helps to save expensive chemicals (specifically CCF4/AM loading kit). In the described workflow it is critical to start image acquisition at a defined time point shortly after addition of the CCF4/AM loading solution because hydrolysis of CCF4 by the translocated beta-lactamase starts immediately. In case of processing to many conditions at once, adjustment of focus and lateral positions (step 5.1.4) may take too long and hamper a timely start of image acquisition. Therefore, the maximum number of parallel samples needs to be determined individually.
One of the main problems encountered using this method was the considerable export of CCF4 substrate by the HeLa cells at 37 &#176;C. This phenomenon could not be sufficiently counteracted by treating the cells with probenecid. We therefore decided, instead of monitoring translocation during the ongoing infection as described previously, to first complete infection and afterwards load the cells with the CCF4/AM dye. With this approach it was possible to monitor the processing of the CCF4 substrate at RT. Under these conditions reduced export of CCF4 was observed. The amount of export of CCF4 substrate seems to be cell line specific; the setup described here allows for reliable analysis of translocation also in cell lines, which strongly export CCF4 at 37 &#176;C.
Export of CCF4 is not a problem in some other cell lines allowing to adopt the microscopy setup for monitoring the processes of infection and translocation simultaneously in real-time. This provides the possibility to analyze translocation into individual cells and could be used to correlate translocation with specific events of host cell interaction like bacterial adhesion or formation of micro-colonies20. Therefore, this assay is a valuable tool to further elucidate the mechanisms of how translocation is regulated during the interplay of bacteria and host cells.

Type III secretion systems are specialized protein-export machines utilized by different genera of gram-negative bacteria to directly deliver bacterially encoded effector proteins into eukaryotic target cells. While the secretion machinery itself is highly conserved, specialized sets of effector proteins have evolved among the different bacterial species to manipulate cellular signaling pathways and facilitate specific bacterial virulence strategies1. In case of Yersinia, up to seven effector proteins, so called Yops (Yersinia outer proteins), are translocated upon host cell contact and act together to subvert immune cell responses such as phagocytosis and cytokine production, i.e., to permit extracellular survival of the bacteria2-4. The process of translocation is tightly controlled at different stages5. It is established that primary activation of the T3SS is triggered by its contact to the target cell6. However, the precise mechanism of this initiation is yet to be elucidated. In Yersinia a second level of so called fine-tuning of translocation is achieved by up- or down-regulating activity of the cellular Rho GTP-binding proteins Rac1 or RhoA. Activation of Rac1 e.g. by invasin or cytotoxic necrotizing factor Y (CNF-Y) leads to increased translocation7-9, while the GAP (GTPase activating protein) function of translocated YopE down-regulates Rac1 activity and accordingly decreases translocation by a negative feedback type of mechanism10,11.
Valid and precise methods are the prerequisite to investigate how translocation is regulated during Yersinia host cell interaction. Many different systems have been used for this purpose, each with specific advantages and drawbacks. Some approaches rely on lysis of infected cells but not bacteria by different detergents followed by western blot analysis. The common drawback of these methods is that minor but inevitable bacterial lysis potentially contaminates the cell lysate with bacteria-associated effector proteins. However, treatment of cells with proteinase K to degrade extracellular effector proteins and subsequent use of digitonin for selective lysis of the eukaryotic cells were proposed to minimize this problem12. Importantly, these assays crucially depend on high quality anti-effector antibodies, which are mostly not commercially available. Attempts to use translational fusions of effector proteins and fluorescent proteins like GFP to monitor translocation were not successful probably due to the globular tertiary structure of the fluorescent proteins and the inability of the secretion apparatus to unfold them before secretion13. However, several different reporter tags like the cya (calmodulin-dependent adenylate cyclase) domain of the Bordetella pertussis toxin cyclolysin14 or the Flash tag were successfully used to analyze translocation. In the former assay the enzymatic activity of cya is used to amplify the signal of the intracellular fusion protein, while the Flash tag, a very short tetracysteine (4Cys) motif tag, allows for labeling with the biarsenical dye Flash without disturbing the process of secretion15.
The approach applied here was reported for the first time by Charpentier et al. and is based on the intracellular conversion of the F&#246;rster resonance energy transfer (FRET) dye CCF4 by translocated effector TEM-1 beta-lactamase fusions16 (Figure&#160;1A). CCF4/AM is a cell-permeant compound in which a coumarin derivate (donor) and a fluorescein moiety (acceptor) are linked by a cephalosporin core. Upon passive entry into the eukaryotic cell, the non-fluorescent esterified CCF4/AM compound is processed by cellular esterases to the charged and fluorescent CCF4 and thereby trapped within the cell. Excitation of the coumarin moiety at 405 nm results in FRET to the fluorescein moiety, which emits a green fluorescence signal at 530 nm. After cleavage of the cephalosporin core by the beta-lactamase FRET is disrupted and excitation of the coumarin moiety leads to blue fluorescence emission at460 nm. Different applications of this method have been described in the literature highlighting its versatility. The method allows for analysis of translocation in vitro and also in in vivo, e.g., the technique was used in a mouse infection model to identify the leukocyte populations targeted for translocation in vivo17-19. Readout of signals can be conducted using plate readers, FACS analysis or fluorescence microscopy. Of note, the method also provides the possibility to monitor translocation in real-time by live-cell microscopy during the infection process20,21. Here laser scanning fluorescence microscopy was applied for readout of fluorescence signals as it provides highest sensitivity and accuracy. In particular, the capability to adjust the emission window with nanometer precision in combination with high-sensitive detectors facilitates optimized fluorescence detection and minimized cross-talk. In addition this microscopy setup can be adapted for real-time monitoring of translocation and potentially permits for simultaneous analysis of host-pathogen interaction at the cellular level.
In this study translocation rates of a Y. enterocolitica wild type strain and a YopE deletion mutant exhibiting a hypertranslocator phenotype10,11 were exemplarily analyzed.

<table><tbody><tr><td>LB-Agar</td><td>Roth</td><td>X969.2</td><td></td></tr><tr><td>LB-Medium</td><td>Roth</td><td>X968.2</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate Buffered Saline</td><td>Sigma-Aldrich</td><td>D8662-500ML</td><td></td></tr><tr><td>96-well plate (black, clear bottom)</td><td>Greiner Bio One</td><td>655087</td><td></td></tr><tr><td>DMEM, high glucose, GlutaMAX Supplement, pyruvate</td><td>Gibco/Life Technologies</td><td>31966-047</td><td></td></tr><tr><td>Probenecid</td><td>Sigma-Aldrich</td><td>P8761-25G</td><td></td></tr><tr><td>LiveBLAzer FRET-B/G Loading Kit with CCF4-AM</td><td>Life Technologies</td><td>K1095</td><td></td></tr><tr><td>Lectin from Triticum vulgaris (wheat) FITC conjugate</td><td>Sigma-Aldrich</td><td>L4895</td><td>Used for peak emission and cross-talk determination</td></tr><tr><td>V450 Rat anti-Mouse CD8a</td><td>BD Bioscience</td><td>560469</td><td>Used for peak emission and cross-talk determination</td></tr><tr><td>Immersol W immersion oil&amp;nbsp;</td><td>Zeiss</td><td>444969-0000-000</td><td>Refractive index = 1.3339 @ 23 &amp;deg;C</td></tr><tr><td>TCS SP5 II confocal laser scanning microscope</td><td>Leica microsystems</td><td></td><td>2x GaAsP-Hybrid detectors, 4 channel spectrometer, acusto optical beam spliter, motorized XY stage, adjustable pinhole, objective 20X HC PL APO CS IMM/CORR, 405&amp;#160;nm diode laser 50&amp;#160;mW</td></tr><tr><td>Imaris 7.6 software</td><td>Bitplane</td><td></td><td>Plugins included ImarisXT and MeasurementPro</td></tr><tr><td>MatLab compiler runtime</td><td>MathWorks</td><td></td><td></td></tr><tr><td>Prism 5</td><td>GraphPad software</td><td></td><td></td></tr></tbody></table>

analysis of yersinia enterocolitica effector translocation into host cells using beta-lactamase effector fusions

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target cells. Inside the host cell the effector proteins manipulate cellular functions to the benefit of the bacteria. To better understand the control of type III secretion during host cell interaction, sensitive and accurate assays to measure translocation are required. We here describe the application of an assay based on the fusion of a Yersinia enterocolitica effector protein fragment (Yersinia outer protein; YopE) with TEM-1 beta-lactamase for quantitative analysis of translocation. The assay relies on cleavage of a cell permeant FRET dye (CCF4/AM) by translocated beta-lactamase fusion. After cleavage of the cephalosporin core of CCF4 by the beta-lactamase, FRET from coumarin to fluorescein is disrupted and excitation of the coumarin moiety leads to blue fluorescence emission. Different applications of this method have been described in the literature highlighting its versatility. The method allows for analysis of translocation in vitro and also in in vivo, e.g., in a mouse model. Detection of the fluorescence signals can be performed using plate readers, FACS analysis or fluorescence microscopy. In the setup described here, in vitro translocation of effector fusions into HeLa cells by different Yersinia mutants is monitored by laser scanning microscopy. Recording intracellular conversion of the FRET reporter by the beta-lactamase effector fusion in real-time provides robust quantitative results. We here show exemplary data, demonstrating increased translocation by a Y. enterocolitica YopE mutant compared to the wild type strain.

To demonstrate the capability of the described method to quantitatively analyze effector translocation into target cells, two Yersinia strains with different translocation kinetics were studied: Y. enterocolitica wild type strain WA-314 (serogroup O8, harboring the virulence plasmid pYVO822) and its derivative WA-314&#916;YopE (WA-314 harboring pYVO8&#916;YopE23). Earlier work showed that Y. enterocolitica mutants lacking functional YopE exhibit a significantly higher Yop translocation rate10. Both strains were transformed with vectors encoding for fusions of the n-terminal secretion signal of YopE and TEM-1 beta-lactamase (pMK-bla)17. WA-314 was additionally transformed with a vector encoding for a respective YopE ovalbumin fusion (pMK-ova17) to serve as a negative control. Incubation times of 30, 60 and 90 min were applied in order to further assess the method`s effective range and suitability for quantification. Two different readouts were used for data analysis: donor fluorescence (Ch1) and relative FRET coefficients (Ch3 = Ch2&#8217; / (Ch1 + Ch2&#8217;)) (Figure 1B and 1C).
For the negative control strain WA-314-pMK-ova plotting of donor channel intensities (Ch1) over time shows no rise of fluorescence emission irrespective of the incubation time (Figure&#160;1B). In contrast, in WA-314-pMK-bla infected cells an increase of fluorescence intensity over time was detected indicating the presence of YopE beta-lactamase fusion in the target cell cytoplasm. As expected the maximum slope of fluorescence intensity is positively correlated with the length of infection (30 min of infection: 0.10/min, 60 min infection: 0.33/min, 90 min infection: 0.76/min) indicating that different concentrations of translocated beta-lactamase can be distinguished. In accordance with previous studies WA-314&#916;YopE-pMK-bla infected cells exhibit more rapid increase of fluorescence intensity compared to the wild type strain (30 min of infection: 1.28/min, 60 min infection: 1.53/min, 90 min infection: 1.41/min). Interestingly, expanding infection to 90 min did not further accelerate the rise of donor fluorescence compared to 60 min of infection (Figure&#160;1B). This finding can presumably be explained by the progressive cytotoxic effect exerted by WA-314&#916;YopE-pMK-bla, which could either inhibit efficient translocation beyond 60 min of infection or lead to loss of fluorescence dyes due to disruption of plasma membrane integrity. The results provided by using the relative FRET coefficient (Ch3) for analysis are in good accordance with the results obtained by the donor channel alone. However, in some conditions (WA-314&#916;YopE-pMK-bla, 60 and 90 min infection) representing very large amounts of translocated beta-lactamase fusion, obviously the CCF4 substrate was instantly processed before imaging could be started. These conditions would not allow for calculating a reasonable maximum negative slope because the relevant part of the curve is missed in this data representation (Figure&#160;1C). Images of the relative FRET coefficient channel provide the possibility to compare differences between individual cells (Figure&#160;1D).
<img alt="Figure 1" src="/files/ftp_upload/53115/53115fig1.jpg" /> 
Figure 1. Quantitative analysis of effector translocation by TEM-1 YopE fusions in Y. enterocolitica. (A) Hydrolysis of CCF4 substrate by translocated TEM-1 beta-lactamase was monitored by laser scanning microscopy. Excitation at 405 nm resulted in green fluorescence emission (530 nm) of the intact substrate and in blue fluorescence emission (460 nm) of the cleaved hydrolysis product (coumarin). (B,&#160;C) Microscopy data were quantitatively analyzed by plotting the donor channel intensity (Ch1) or calculation and plotting of the relative FRET coefficients (Ch3 = Ch2&#8217; / (Ch1 + Ch2&#8217;)). Data are representative of three independent experiments. (D) Depicted micrographs (FRET channel) were taken from representative movies at the indicated time points. WA-314&#916;E-pMK-bla infected cells show more rapid decrease of the relative FRET coefficients compared to the wild type WA-314-pMK-bla (60 min infection). Scale bar, 20 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53115/53115fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53115/53115fig2.jpg" /> 
Figure 2. Cross-talk determination of the fluorophores V450 and FITC. The spectra of both individual fluorophores were measured with the confocal microscope. Bleed-through of the donor V450 into the acceptor channel within the 525-535 nm detector range was calculated from a linear regression (insert). The mean percentage of crosstalk calculated from two measurements equals 0.33. Bleed-through of the acceptor FITC into the donor channel (455-465 nm) was not detectable over the level of noise. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53115/53115fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions at JoVE.com

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Effector translocation into host cells via a type III secretion system is a common virulence strategy among gram-negative bacteria. A beta-lactamase effector fusion based assay for quantitative analysis of translocation was applied. In Yersinia infected cells, conversion of a FRET reporter by the beta-lactamase is monitored using laser scanning microscopy.

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target ...

analysis-yersinia-enterocolitica-effector-translocation-into-host

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

8.5K Views.  University Medical Center Hamburg-Eppendorf. Effector translocation into host cells via a type III secretion system is a common virulence strategy among gram-negative bacteria. A beta-lactamase effector fusion based assay for quantitative analysis of translocation was applied. In Yersinia infected cells, conversion of a FRET reporter by the beta-lactamase is monitored using laser scanning microscopy.

Video: Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed compartment allows bacteria to move within the cytosol, proliferate and further invade neighboring cells. Mycobacterium tuberculosis is phagocytosed by immune cells, and has recently been shown to rupture phagosomal membrane in macrophages. We developed a robust assay for tracking phagosomal membrane disruption after host cell entry of Shigella flexneri or Mycobacterium tuberculosis. The approach makes use of CCF4, a FRET reporter sensitive to &beta;-lactamase that equilibrates in the cytosol of host cells. Upon invasion of host cells by bacterial pathogens, the probe remains intact as long as the bacteria reside in membrane-enclosed compartments. After disruption of the vacuole, &beta;-lactamase activity on the surface of the intracellular pathogen cleaves CCF4 instantly leading to a loss of FRET signal and switching its emission spectrum. This robust ratiometric assay yields accurate information about the timing of vacuolar rupture induced by the invading bacteria, and it can be coupled to automated microscopy and image processing by specialized algorithms for the detection of the emission signals of the FRET donor and acceptor. Further, it allows investigating the dynamics of vacuolar disruption elicited by intracellular bacteria in real time in single cells. Finally, it is perfectly suited for high-throughput analysis with a spatio-temporal resolution exceeding previous methods. Here, we provide the experimental details of exemplary protocols for the CCF4 vacuolar rupture assay on HeLa cells and THP-1 macrophages for time-lapse experiments or end points experiments using Shigella flexneri as well as multiple mycobacterial strains such as Mycobacterium marinum, Mycobacterium bovis, and Mycobacterium tuberculosis.

We describe a method for tracking the endomembrane rupture elicited by the intracellular bacteria Shigella flexneri and Mycobacterium tuberculosis upon host cell invasion. Our assay makes use of CCF4, a host cytoplasmic FRET probe in live or fixed cells. This reporter is degraded by an enzyme activity present on the bacterial surface.

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed ...

Identification of Host Pathways Targeted by Bacterial Effector Proteins using Yeast Toxicity and Suppressor Screens

Intracellular bacteria secrete virulence factors called effector proteins into the host cytosol that act to subvert host proteins and/or their associated biological pathways to the benefit of the bacterium. Identification of putative bacterial effector proteins has become more manageable due to advances in bacterial genome sequencing and the advent of algorithms that allow in silico identification of genes encoding secretion candidates and/or eukaryotic-like domains. However, identification of these important virulence factors is only an initial step. Naturally, the goal is to determine the molecular function of effector proteins and elucidate how they interact with the host. In recent years, techniques like the yeast two-hybrid screen and large-scale immunoprecipitations coupled with mass spectrometry have aided in the identification of protein-protein interactions. Although identification of a host binding partner is the crucial first step toward elucidating the molecular function of a bacterial effector protein, sometimes the host protein is found to have multiple biological functions (e.g., actin, clathrin, tubulin), or the bacterial protein may not physically bind host proteins, depriving the researcher of crucial information about the precise host pathway being manipulated. A modified yeast toxicity screen coupled with a suppressor screen has been adapted to identify host pathways impacted by bacterial effector proteins. The toxicity screen relies on a toxic effect in yeast caused by the effector protein interfering with the host biological pathways, which often manifests as a growth defect. Expression of a yeast genomic library is used to identify host factors that suppress the toxicity of the bacterial effector protein and thus identify proteins in the pathway that the effector protein targets. This protocol contains detailed instructions for both the toxicity and suppressor screens. These techniques can be performed in any lab capable of molecular cloning and cultivation of yeast and Escherichia coli.

Bacterial pathogens secrete proteins into the host that target crucial biological processes. Identifying the host pathways targeted by bacterial effector proteins is key to addressing molecular pathogenesis. Here, a method using a modified yeast suppressor and toxicity screen to elucidate host pathways targeted by toxic bacterial effector proteins is described.

Intracellular bacteria secrete virulence factors called effector proteins into the host cytosol that act to subvert host proteins and/or their associated ...

Genetics

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 ...

Electroporation of Functional Bacterial Effectors into Mammalian Cells

The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. This information is particularly valuable in the context of host-pathogen interactions. Many pathogen proteins function within host cells in a variety of way such as, enabling evasion of the host immune system and survival within the intracellular environment. To study these pathogen-protein host-cell interactions, several approaches are commonly used, including: in vivo infection with a strain expressing a tagged or mutant protein, or introduction of pathogen genes via transfection or transduction. Each of these approaches has advantages and disadvantages. We sought a means to directly introduce exogenous proteins into cells. Electroporation is commonly used to introduce nucleic acids into cells, but has been more rarely applied to proteins although the biophysical basis is exactly the same. A standard electroporator was used to introduce affinity-tagged bacterial effectors into mammalian cells. Human epithelial and mouse macrophage cells were cultured by traditional methods, detached, and placed in 0.4 cm gap electroporation cuvettes with an exogenous bacterial pathogen protein of interest (e.g. Salmonella Typhimurium GtgE). After electroporation (0.3 kV) and a short (4 hr) recovery period, intracellular protein was verified by fluorescently labeling the protein via its affinity tag and examining spatial and temporal distribution by confocal microscopy. The electroporated protein was also shown to be functional inside the cell and capable of correct subcellular trafficking and protein-protein interaction. While the exogenous proteins tended to accumulate on the surface of the cells, the electroporated samples had large increases in intracellular effector concentration relative to incubation alone. The protocol is simple and fast enough to be done in a parallel fashion, allowing for high-throughput characterization of pathogen proteins in host cells including subcellular targeting and function of virulence proteins.

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.

The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. ...

Identification of Growth Inhibition Phenotypes Induced by Expression of Bacterial Type III Effectors in Yeast

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within the cell, type III effectors subvert host cellular processes to suppress immune responses and promote pathogen growth. Numerous type III effectors of plant and animal bacterial pathogens have been identified to date, yet only a few of them are well characterized. Understanding the functions of these effectors has been undermined by a combination of functional redundancy in the effector repertoire of a given bacterial strain, the subtle effects that they may exert to increase virulence, roles that are possibly specific to certain infection stages, and difficulties in genetically manipulating certain pathogens. Expression of type III effectors in the budding yeast Saccharomyces cerevisiae may allow circumventing these limitations and aid to the functional characterization of effector proteins. Because type III effectors often target cellular processes that are conserved between yeast and other eukaryotes, their expression in yeast may result in growth inhibition phenotypes that can be exploited to elucidate effector functions and targets. Additional advantages to using yeast for functional studies of bacterial effectors include their genetic tractability, information on predicted functions of the vast majority of their ORFs, and availability of numerous tools and resources for both genome-wide and small-scale experiments. Here we discuss critical factors for designing a yeast system for the expression of bacterial type III effector proteins. These include an appropriate promoter for driving expression of the effector gene(s) of interest, the copy number of the effector gene, the epitope tag used to verify protein expression, and the yeast strain. We present procedures to induce expression of effectors in yeast and to verify their expression by immunoblotting. In addition, we describe a spotting assay on agar plates for the identification of effector-induced growth inhibition phenotypes. The use of this protocol may be extended to the study of pathogenicity factors delivered into the host cell by any pathogen and translocation mechanism.

In this video, we describe a procedure for the expression of bacterial type III effectors in yeast and the identification of effector-induced growth inhibition phenotypes. Such phenotypes can be subsequently exploited to elucidate effector functions and targets.

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within ...

Biology

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 ...

Unravelling the Function of a Bacterial Effector from a Non-cultivable Plant Pathogen Using a Yeast Two-hybrid Screen

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria have evolved different strategies to manipulate their host metabolism for their own benefit. This bacterial manipulation is often coupled with severe symptom development or the death of the affected plants. Determining the specific bacterial molecules responsible for the host manipulation has become an important field in microbiological research. After the identification of these bacterial molecules, called &#34;effectors,&#34; it is important to elucidate their function. A straightforward approach to determine the function of an effector is to identify its proteinaceous binding partner in its natural host via a yeast two-hybrid (Y2H) screen. Normally the host harbors numerous potential binding partners that cannot be predicted sufficiently by any in silico algorithm. It is thus the best choice to perform a screen with the hypothetical effector against a whole library of expressed host proteins. It is especially challenging if the causative agent is uncultivable like phytoplasma. This protocol provides step-by-step instructions for DNA purification from a phytoplasma-infected woody host plant, the amplification of the potential effector, and the subsequent identification of the plant&#39;s molecular interaction partner with a Y2H screen. Even though Y2H screens are commonly used, there is a trend to outsource this technique to biotech companies that offer the Y2H service at a cost. This protocol provides instructions on how to perform a Y2H in any decently equipped molecular biology laboratory using standard lab techniques.

Bacterial effector proteins are important for establishing successful infections. This protocol describes the experimental identification of proteinaceous binding partners of a bacterial effector protein in its natural plant host. Identifying these effector interactions via yeast two-hybrid screens has become an important tool in unravelling molecular pathogenicity strategies.

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria ...

Generation of Enterobacter sp. YSU Auxotrophs Using Transposon Mutagenesis

Prototrophic bacteria grow on M-9 minimal salts medium supplemented with glucose (M-9 medium), which is used as a carbon and energy source. Auxotrophs can be generated using a transposome. The commercially available, Tn5-derived transposome used in this protocol consists of a linear segment of DNA containing an R6K&#947; replication origin, a gene for kanamycin resistance and two mosaic sequence ends, which serve as transposase binding sites. The transposome, provided as a DNA/transposase protein complex, is introduced by electroporation into the prototrophic strain, Enterobacter sp. YSU, and randomly incorporates itself into this host&#8217;s genome. Transformants are replica plated onto Luria-Bertani agar plates containing kanamycin, (LB-kan) and onto M-9 medium agar plates containing kanamycin (M-9-kan). The transformants that grow on LB-kan plates but not on M-9-kan plates are considered to be auxotrophs. Purified genomic DNA from an auxotroph is partially digested, ligated and transformed into a pir+ Escherichia coli (E. coli) strain. The R6K&#947; replication origin allows the plasmid to replicate in pir+ E. coli strains, and the kanamycin resistance marker allows for plasmid selection. Each transformant possesses a new plasmid containing the transposon flanked by the interrupted chromosomal region. Sanger sequencing and the Basic Local Alignment Search Tool (BLAST) suggest a putative identity of the interrupted gene. There are three advantages to using this transposome mutagenesis strategy. First, it does not rely on the expression of a transposase gene by the host. Second, the transposome is introduced into the target host by electroporation, rather than by conjugation or by transduction and therefore is more efficient. Third, the R6K&#947; replication origin makes it easy to identify the mutated gene which is partially recovered in a recombinant plasmid. This technique can be used to investigate the genes involved in other characteristics of Enterobacter sp. YSU or of a wider variety of bacterial strains.

Generation of Enterobacter sp. YSU Auxotrophs Using Transposon Mutagenesis

Enterobacter sp. YSU grows in glucose minimal salts medium. Auxotrophs are generated by transforming it with a transposome which randomly inserts itself into the host genome. Mutants are found by replica plating from complex medium to minimal medium. Interrupted genes are identified by gene rescue and sequencing.

Prototrophic bacteria grow on M-9 minimal salts medium supplemented with glucose (M-9 medium), which is used as a carbon and energy source. Auxotrophs can ...

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 ...

Super-Resolution Imaging of Bacterial Secreted Proteins Using Genetic Code Expansion

Type three secretion systems (T3SSs) enable gram-negative bacteria to inject a battery of effector proteins directly into the cytosol of eukaryotic host cells. Upon entry, the injected effector proteins cooperatively modulate eukaryotic signaling pathways and reprogram cellular functions, enabling bacterial entry and survival. Monitoring and localizing these secreted effector proteins in the context of infections provides a footprint for defining the dynamic interface of host-pathogen interactions. However, labeling and imaging bacterial proteins in host cells without disrupting their structure/function is technically challenging. 
Constructing fluorescent fusion proteins does not resolve this problem, because the fusion proteins jam the secretory apparatus and thus are not secreted. To overcome these obstacles, we recently employed a method for site-specific fluorescent labeling of bacterial secreted effectors, as well as other difficult-to-label proteins, using genetic code expansion (GCE). This paper provides a complete step-by-step protocol to label Salmonella secreted effectors using GCE site-specifically, followed by directions for imaging the subcellular localization of secreted proteins in HeLa cells using direct stochastic optical reconstruction microscopy (dSTORM) 
Recent findings suggest that the incorporation of non-canonical amino acids (ncAAs) via GCE, followed by bio-orthogonal labeling with tetrazine-containing dyes, is a viable technique for selective labeling and visualization of bacterial secreted proteins and subsequent image analysis in the host. The goal of this article is to provide a straightforward and clear protocol that can be employed by investigators interested in conducting super-resolution imaging using GCE to study various biological processes in bacteria and viruses, as well as host-pathogen interactions.

This article provides a straightforward and clear protocol to label Salmonella secreted effectors using genetic code expansion (GCE) site-specifically and image the subcellular localization of secreted proteins in HeLa cells using direct stochastic optical reconstruction microscopy (dSTORM)

Type three secretion systems (T3SSs) enable gram-negative bacteria to inject a battery of effector proteins directly into the cytosol of eukaryotic host ...

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

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