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>

<ol>
	<li>Galan, J. E., Lara-Tejero, M., Marlovits, T. C., Wagner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+Type+III+Secretion+Systems:+Specialized+Nanomachines+for+Protein+Delivery+into+Target+Cells.">Bacterial Type III Secretion Systems: Specialized Nanomachines for Protein Delivery into Target Cells.</a> Annu Rev Microbiol. 68, 415-438 (2014).</li><li>Aepfelbacher, M., Trasak, C., Ruckdeschel, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effector+functions+of+pathogenic+Yersinia+species.">Effector functions of pathogenic Yersinia species.</a> Thromb Haemost. 98 (3), 521-529 (2007).</li><li>Heesemann, J., Sing, A., Trulzsch, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yersinia's+stratagem:+targeting+innate+and+adaptive+immune+defense.">Yersinia's stratagem: targeting innate and adaptive immune defense.</a> Curr Opin Microbiol. 9 (1), 55-61 (2006).</li><li>Viboud, G. I., Bliska, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yersinia+outer+proteins:+role+in+modulation+of+host+cell+signaling+responses+and+pathogenesis.">Yersinia outer proteins: role in modulation of host cell signaling responses and pathogenesis.</a> Annu Rev Microbiol. 59, 69-89 (2005).</li><li>Dewoody, R. S., Merritt, P. M., Marketon, M. 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B., Viboud, G. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yersinia+controls+type+III+effector+delivery+into+host+cells+by+modulating+Rho+activity.">Yersinia controls type III effector delivery into host cells by modulating Rho activity.</a> PLoS Pathog. 4 (1), e3 (2008).</li><li>Aili, 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=Regulation+of+Yersinia+Yop-effector+delivery+by+translocated+YopE.">Regulation of Yersinia Yop-effector delivery by translocated YopE.</a> Int J Med Microbiol. 298 (3-4), 183-192 (2008).</li><li>Gaus, K., 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=Destabilization+of+YopE+by+the+ubiquitin-proteasome+pathway+fine-tunes+Yop+delivery+into+host+cells+and+facilitates+systemic+spread+of+Yersinia+enterocolitica+in+host+lymphoid+tissue.">Destabilization of YopE by the ubiquitin-proteasome pathway fine-tunes Yop delivery into host cells and facilitates systemic spread of Yersinia enterocolitica in host lymphoid tissue.</a> Infect Immun. 79 (3), 1166-1175 (2011).</li><li>Nordfelth, R., Wolf-Watz, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=YopB+of+Yersinia+enterocolitica+is+essential+for+YopE+translocation.">YopB of Yersinia enterocolitica is essential for YopE translocation.</a> Infect Immun. 69 (5), 3516-3518 (2001).</li><li>Radics, J., Konigsmaier, L., Marlovits, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+a+pathogenic+type+3+secretion+system+in+action.">Structure of a pathogenic type 3 secretion system in action.</a> Nat Struct Mol Biol. 21 (1), 82-87 (2014).</li><li>Sory, M. P., Cornelis, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translocation+of+a+hybrid+YopE-adenylate+cyclase+from+Yersinia+enterocolitica+into+HeLa+cells.">Translocation of a hybrid YopE-adenylate cyclase from Yersinia enterocolitica into HeLa cells.</a> Mol Microbiol. 14 (3), 583-594 (1994).</li><li>Schlumberger, M. 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. M., DePaolo, R. W., DeBord, K. L., Jabri, B., Schneewind, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plague+bacteria+target+immune+cells+during+infection.">Plague bacteria target immune cells during infection.</a> Science. 309 (5741), 1739-1741 (2005).</li><li>Mills, E., Baruch, K., Aviv, G., Nitzan, M., Rosenshine, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+the+type+III+secretion+system+activity+of+enteropathogenic+Escherichia+coli.">Dynamics of the type III secretion system activity of enteropathogenic Escherichia coli.</a> MBio. 4 (4), (2013).</li><li>Mills, E., Baruch, K., Charpentier, X., Kobi, S., Rosenshine, I. <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+analysis+of+effector+translocation+by+the+type+III+secretion+system+of+enteropathogenic+Escherichia+coli.">Real-time analysis of effector translocation by the type III secretion system of enteropathogenic Escherichia coli.</a> Cell Host Microbe. 3 (2), 104-113 (2008).</li><li>Heesemann, J., Laufs, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+a+mobilizable+Yersinia+enterocolitica+virulence+plasmid.">Construction of a mobilizable Yersinia enterocolitica virulence plasmid.</a> J Bacteriol. 155 (2), 761-767 (1983).</li><li>Zumbihl, R., 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=The+cytotoxin+YopT+of+Yersinia+enterocolitica+induces+modification+and+cellular+redistribution+of+the+small+GTP-binding+protein+RhoA.">The cytotoxin YopT of Yersinia enterocolitica induces modification and cellular redistribution of the small GTP-binding protein RhoA.</a> J Biol Chem. 274 (41), 29289-29293 (1999).</li></ol>

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 detec...

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 border="0" cellpadding="0" cellspacing="0" width="1068">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">LB-Agar</td>
			<td style="width:209px;">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&#8217;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&#160;</td>
			<td>Zeiss</td>
			<td>444969-0000-000</td>
			<td>Refractive index = 1.3339 @ 23 &#176;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, 405nm diode laser 50mW</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 ...

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

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.