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 a...

<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. M. <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+the+Yersinia+type+III+secretion+system:+traffic+control.">Regulation of the Yersinia type III secretion system: traffic control.</a> Front Cell Infect Microbiol. 3, 10-3389 (2013).</li><li>Pettersson, J., 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=Modulation+of+virulence+factor+expression+by+pathogen+target+cell+contact.">Modulation of virulence factor expression by pathogen target cell contact.</a> Science. 273 (5279), 1231-1233 (1996).</li><li>Schweer, J., 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+cytotoxic+necrotizing+factor+of+Yersinia+pseudotuberculosis+(CNFY)+enhances+inflammation+and+Yop+delivery+during+infection+by+activation+of+Rho+GTPases.">The cytotoxic necrotizing factor of Yersinia pseudotuberculosis (CNFY) enhances inflammation and Yop delivery during infection by activation of Rho GTPases.</a> PLoS Pathog. 9 (11), e1003746 (2013).</li><li>Wolters, 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=Cytotoxic+necrotizing+factor-Y+boosts+Yersinia+effector+translocation+by+activating+Rac+protein.">Cytotoxic necrotizing factor-Y boosts Yersinia effector translocation by activating Rac protein.</a> J Biol Chem. 288 (32), 23543-23553 (2013).</li><li>Mejia, E., Bliska, J. 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>

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

8.4K 次观看.  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.

视频: 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...

introduction of bacterial effector proteins into mammalian cells by electroporation

In this video, we demonstrate the introduction of bacterial effector proteins into mammalian cells by electroporation. The intracellular localization of the bacterial effector proteins is detected by confocal microscopy following staining with specific fluorophore-conjugated...

Introduction of Bacterial Effector Proteins into Mammalian Cells by Electroporation

Place the sample in the electroporator and set it to 0.3 kilovolts for 1.5 to 1.7 milliseconds. Immediately after electroporation, flick the cuvette gently, about 10 times, to mix the sample thoroughly before proceeding to either fixation and immunofluorescence staining or affinity...

applying fluorescence resonance energy transfer (fret) to examine effector translocation efficiency by coxiella burnetii during sirna silencing

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...

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

identification of host pathways targeted by bacterial effector proteins using yeast toxicity and suppressor screens

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

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...

delivering effector t lymphocytes into mice carrying human brain tumors

This video demonstrates a stereotaxic injection technique for administering human effector T lymphocytes into a murine model carrying a human brain tumor. Upon positioning a tumor-bearing anesthetized mouse on a stereotactic frame and creating a burr hole at the injection location on the skull, effector T lymphocytes are injected at the tumor...

Delivering Effector T Lymphocytes into Mice Carrying Human Brain Tumors

Then, place the syringe into the adapted syringe pump. Disinfect the surgical site with a swab soaked in povidone-iodine 5% solution, and place a lubricating ophthalmic ointment in the mouse&#39;s eyes to prevent drying of the cornea. Then, position the anesthetized mouse on the stereotactic frame on a warm isothermal block. Appropriately position mouse&#39;s nose and teeth above the tooth bar. Tighten ear bars firmly in the mouse&#39;s ears to immobilize the head. Be careful not to damage the...

induction of murine intestinal inflammation by adoptive transfer of effector cd4+cd45rbhigh t cells into immunodeficient mice

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

detection of toxin translocation into the host cytosol by surface plasmon resonance

Detection of Toxin Translocation into the Host Cytosol by Surface Plasmon Resonance

In this report, we describe how surface plasmon resonance is used to detect toxin entry into the host cytosol.  This highly sensitive method can provide quantitative data on the amount of cytosolic toxin, and it can be applied to a range of...

imaging effector memory t cells in the ear after induction of adoptive dth

Imaging Effector Memory T cells in the Ear After Induction of Adoptive DTH

Here we demonstrate a method for inducing and recording the progress of a delayed type-hypersensitivity (DTH) reaction in the rat ear. This is followed by a demonstration of the preparation of rat ear tissue for two-photon imaging of the effector / memory T cell...

quantifying yersinia pseudotuberculosis type iii secretion system activity following iron starvation and anaerobic growth

Author Spotlight: Molecular Mechanisms Underlying T3SS Expression Under Variable Iron and Oxygen Availability  

Bacteria colonize host tissues that vary in oxygen and iron bioavailability, yet most approaches to studying bacteria use aerated, rich media. This protocol describes culturing the human pathogen Yersinia pseudotuberculosis under varying iron concentrations and oxygen tension, and quantifying activity of the Yersinia type III secretion system, which is an important virulence factor.

T3SS Activity in &lt;i&gt;Yersinia pseudotuberculosis&lt;/i&gt;  Under Iron Depletion &amp; Anaerobic Stress 

https://cloudfront.jove.com/CDNSource/teasers/66642_b.jpg

genome editing in astyanax mexicanus using transcription activator-like effector nucleases (talens)

Genome Editing in Astyanax mexicanus Using Transcription Activator-like Effector Nucleases TALENs

Gene-targeting mutagenesis is now possible in a wide range of organisms using genome editing techniques. Here, we demonstrate a protocol for targeted gene mutagenesis using transcription activator like effector nucleases (TALENs) in Astyanax mexicanus, a species of fish that includes surface fish and...

Genome Editing in Astyanax mexicanus Using Transcription Activator-like Effector Nucleases (TALENs)

measuring the activation of fc-mediated effector functions by ha antibodies

This video demonstrates an assay measuring the antibody-mediated activation of T cell effector functions. Adding an influenza virus hemagglutinin (HA)-specific monoclonal antibody to transfected mammalian cells expressing HA results in the formation of immune complexes with HA. Upon introducing engineered T cells expressing Fc receptors and a luciferase reporter, the T cells are activated by the antibody-HA complexes, and this activation is detected by adding a luciferase substrate and...

Measuring the Activation of Fc-Mediated Effector Functions by HA Antibodies

Next, in a separate 96-well plate, perform 4-fold dilutions of the antibodies of interest in triplicate and fresh ADCC medium starting at 10 micrograms per milliliter according to the schematic, and taking care to include the background and no antibody...

detection of signaling effector-complexes downstream of bmp4 using in situ pla, a proximity ligation assay

Detection of Signaling Effector-Complexes Downstream of BMP4 Using in situ PLA, a Proximity Ligation Assay

Here we show how to use Proximity Ligation Assay (PLA), with a combination of antibodies to visualize Bone Morphogenetic Protein (BMP) signaling in fixed cells. This technique allowed us to follow the nuclear accumulation of endogenous BMP activated effector-complexes and quantify their levels over time under BMP4...

Detection of Signaling Effector-Complexes Downstream of BMP4 Using in situ PLA, a Proximity Ligation Assay

measuring peptide translocation into large unilamellar vesicles

Measuring Peptide Translocation into Large Unilamellar Vesicles

This protocol details a method for the quantitative measure of peptide translocation into large unilamellar lipid vesicles.  This method also provides information about the rate of membrane translocation and can be used to identify peptides that efficiently and spontaneously cross lipid...

unravelling the function of a bacterial effector from a non-cultivable plant pathogen using a yeast two-hybrid screen

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

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...

a chronic autoimmune dry eye rat model with increase in effector memory t cells in eyeball tissue

A Chronic Autoimmune Dry Eye Rat Model with Increase in Effector Memory T Cells in Eyeball Tissue

This report describes a method to induce chronic experimental autoimmune dry eye in Lewis rats through immunization with an emulsion of rat lacrimal gland extract, ovalbumin, and complete Freund's adjuvant, followed by the injection of lacrimal gland extract and ovalbumin into the forniceal subconjunctiva and lacrimal glands six weeks...

capture compound mass spectrometry - a powerful tool to identify novel c-di-gmp effector proteins

Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

The ubiquitous second messenger c-di-GMP controls growth and behavior of many bacteria. We have developed a novel Capture Compound Mass Spectrometry based technology to biochemically identify and characterize c-di-GMP binding proteins in virtually any bacterial...

stereotaxic infusion of oligomeric amyloid-beta into the mouse hippocampus

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

Here, we present a protocol for direct stereotaxic brain infusion of amyloid-beta. This methodology provides an alternative in vivo mouse model to address the short-term effects of amyloid-beta on brain...

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

Viral pathogens represent a significant public health threat; not only can viruses cause natural epidemics of human disease, but their potential use in bioterrorism is also a concern. A better understanding of the cellular factors that impact infection would facilitate the development of much-needed therapeutics. Recent advances in RNA interference (RNAi) technology coupled with complete genome sequencing of several organisms has led to the optimization of genome-wide, cell-based loss-of-function screens. Drosophila cells are particularly amenable to genome-scale screens because of the ease and efficiency of RNAi in this system 1. Importantly, a wide variety of viruses can infect Drosophila cells, including a number of mammalian viruses of medical and agricultural importance 2,3,4. Previous RNAi screens in Drosophila have identified host factors that are required for various steps in virus infection including entry, translation and RNA replication 5. Moreover, many of the cellular factors required for viral replication in Drosophila cell culture are also limiting in human cells infected with these viruses 4,6,7,8, 9. Therefore, the identification of host factors co-opted during viral infection presents novel targets for antiviral therapeutics. Here we present a generalized protocol for a high-throughput RNAi screen to identify cellular factors involved in viral infection, using vaccinia virus as an example.

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

Novel host factors involved in viral infection can be identified through cell-based genome-wide loss of function RNAi screening. A Drosophila cell culture model is particularly amenable to this approach due to the ease and efficiency of RNAi. Here we demonstrate this technique using vaccinia virus as an example.

Viral pathogens represent a significant public health threat; not only can viruses cause natural epidemics of human disease, but their potential use in ...

AB toxins consist of an enzymatic A subunit and a cell-binding B subunit1. These toxins are secreted into the extracellular milieu, but they act upon targets within the eukaryotic cytosol. Some AB toxins travel by vesicle carriers from the cell surface to the endoplasmic reticulum (ER) before entering the cytosol2-4. In the ER, the catalytic A chain dissociates from the rest of the toxin and moves through a protein-conducting channel to reach its cytosolic target5. The translocated, cytosolic A chain is difficult to detect because toxin trafficking to the ER is an extremely inefficient process: most internalized toxin is routed to the lysosomes for degradation, so only a small fraction of surface-bound toxin reaches the Golgi apparatus and ER6-12.

To monitor toxin translocation from the ER to the cytosol in cultured cells, we combined a subcellular fractionation protocol with the highly sensitive detection method of surface plasmon resonance (SPR)13-15. The plasma membrane of toxin-treated cells is selectively permeabilized with digitonin, allowing collection of a cytosolic fraction which is subsequently perfused over an SPR sensor coated with an anti-toxin A chain antibody. The antibody-coated sensor can capture and detect pg/mL quantities of cytosolic toxin. With this protocol, it is possible to follow the kinetics of toxin entry into the cytosol and to characterize inhibitory effects on the translocation event. The concentration of cytosolic toxin can also be calculated from a standard curve generated with known quantities of A chain standards that have been perfused over the sensor. Our method represents a rapid, sensitive, and quantitative detection system that does not require radiolabeling or other modifications to the target toxin.

In this report, we describe how surface plasmon resonance is used to detect toxin entry into the host cytosol. This highly sensitive method can provide quantitative data on the amount of cytosolic toxin, and it can be applied to a range of toxins.

AB toxins consist of an enzymatic A subunit and a cell-binding B subunit1.  These toxins are secreted into the extracellular milieu, but they act upon ...

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are upregulated upon this ongoing antigen exposure and contribute to loss of proliferation, reduced cytolytic function, and impaired cytokine production by CD4 and CD8 T cells. In the murine model of LCMV infection, administration of blocking antibodies against these two pathways augmented T cell responses. However, there is currently no in vitro assay to measure the impact of such blockade on cytokine secretion in cells from human samples. Our protocol and experimental approach enable us to accurately and efficiently quantify the restoration of cytokine production by HIV-specific CD4 T cells from HIV infected subjects.
Here, we depict an in vitro experimental design that enables measurements of cytokine secretion by HIV-specific CD4 T cells and their impact on other cell subsets. CD8 T cells were depleted from whole blood and remaining PBMCs were isolated via Ficoll separation method. CD8-depleted PBMCs were then incubated with blocking antibodies against PD-L1 and/or IL-10R&#945; and, after stimulation with an HIV-1 Gag peptide pool, cells were incubated at 37 &#176;C, 5% CO2. After 48 hr, supernatant was collected for cytokine analysis by beads arrays and cell pellets were collected for either phenotypic analysis using flow cytometry or transcriptional analysis using qRT-PCR. For more detailed analysis, different cell populations were obtained by selective subset depletion from PBMCs or by sorting using flow cytometry before being assessed in the same assays. These methods provide a highly sensitive and specific approach to determine the modulation of cytokine production by antigen-specific T-helper cells and to determine functional interactions between different populations of immune cells.

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

We developed an in vitro assay to investigate the role of immunoregulatory pathways in the regulation of cytokine secretion by HIV-specific CD4 T cells.

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are ...

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

Application of Long-term cultured Interferon-&#947; Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is controlled up to 95 % of the T cells generated during the expansion phase are eliminated (i.e., contraction phase) and memory T cells remain, sometimes for a lifetime. In humans, two functionally distinct subsets of memory T cells have been described based on the expression of lymph node homing receptors. Central memory T cells express C-C chemokine receptor 7 and CD45RO and are mainly located in T-cell areas of secondary lymphoid organs. Effector memory T cells express CD45RO, lack CCR7 and display receptors associated with lymphocyte homing to peripheral or inflamed tissues. Effector T cells do not express either CCR7 or CD45RO but upon encounter with antigen produce effector cytokines, such as interferon-&#947;. Interferon-&#947; release assays are used for the diagnosis of bovine and human tuberculosis and detect primarily effector and effector memory T cell responses. Central memory T cell responses by CD4+ T cells to vaccination, on the other hand, may be used to predict vaccine efficacy, as demonstrated with simian immunodeficiency virus infection of non-human primates, tuberculosis in mice, and malaria in humans. Several studies with mice and humans as well as unpublished data on cattle, have demonstrated that interferon-&#947; ELISPOT assays measure central memory T cell responses. With this assay, peripheral blood mononuclear cells are cultured in decreasing concentration of antigen for 10 to 14 days (long-term culture), allowing effector responses to peak and wane; facilitating central memory T cells to differentiate and expand within the culture.

Application of Long-term cultured Interferon-&amp;#947; Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle

Long-term cultured interferon-&#947; enzyme-linked immunospot assay is used as a measure of central memory responses and correlates with protective anti-mycobacterial vaccine responses. With this assay, peripheral blood mononuclear cells are stimulated with mycobacterial antigens and interleukin-2 for 14 days, enabling differentiation and expansion of central memory T cells.

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is ...

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.

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 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 ...

Dry eye disease is a very common condition that causes morbidity and healthcare burden and decreases the quality of life. There is a need for a suitable dry eye animal model to test novel therapeutics to treat autoimmune dry eye conditions. This protocol describes a chronic autoimmune dry eye rat model. Lewis rats were immunized with an emulsion containing lacrimal gland extract, ovalbumin, and complete Freund&#39;s adjuvant. A second immunization with the same antigens in incomplete Freund&#39;s adjuvant was administered two weeks later. These immunizations were administered subcutaneously at the base of the tail. To boost the immune response at the ocular surface and lacrimal glands, lacrimal gland extract and ovalbumin were injected into the forniceal subconjunctiva and lacrimal glands 6 weeks after the first immunization. The rats developed dry eye features, including reduced tear production, decreased tear stability, and increased corneal damage. Immune profiling by flow cytometry showed a preponderance of CD3+ effector memory T cells in the eyeball.

This report describes a method to induce chronic experimental autoimmune dry eye in Lewis rats through immunization with an emulsion of rat lacrimal gland extract, ovalbumin, and complete Freund's adjuvant, followed by the injection of lacrimal gland extract and ovalbumin into the forniceal subconjunctiva and lacrimal glands six weeks later.

Dry eye disease is a very common condition that causes morbidity and healthcare burden and decreases the quality of life. There is a need for a suitable ...

A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and Fc-regions. Here, we describe how to measure the activation of Fc effector functions by monoclonal antibodies targeting the influenza virus hemagglutinin with the use of a genetically engineered Jurkat cell line expressing an activating type 1 Fc-Fc&#947;R. Using this method, the contribution of specific Fc-Fc&#947;R interactions conferred by immunoglobulins can be determined using an in vitro assay.

We describe a method to measure the activation of Fc-mediated effector functions by antibodies that target the influenza virus hemagglutinin. This assay can also be adapted to assess the ability of monoclonal antibodies or polyclonal sera targeting other viral surface glycoproteins to induce Fc-mediated immunity.

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and ...

Temporal Analysis of the Nuclear-to-cytoplasmic Translocation of a Herpes Simplex Virus 1 Protein by Immunofluorescent Confocal Microscopy

Infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) is an immediate early protein containing a RING-type E3 ubiquitin ligase. It is responsible for the proteasomal degradation of host restrictive factors and the subsequent viral gene activation. ICP0 contains a canonical nuclear localization sequence (NLS). It enters the nucleus immediately after de novo synthesis and executes its anti-host defense functions mainly in the nucleus. However, later in infection, ICP0 is found solely in the cytoplasm, suggesting the occurrence of a nuclear-to-cytoplasmic translocation during HSV-1 infection. Presumably ICP0 translocation enables ICP0 to modulate its functions according to its subcellular locations at different infection phases. In order to delineate the biological function and regulatory mechanism of ICP0 nuclear-to-cytoplasmic translocation, we modified an immunofluorescent microscopy method to monitor ICP0 trafficking during HSV-1 infection. This protocol involves immunofluorescent staining, confocal microscope imaging, and nuclear vs. cytoplasmic distribution analysis. The goal of this protocol is to adapt the steady state confocal images taken in a time course into a quantitative documentation of ICP0 movement throughout the lytic infection. We propose that this method can be generalized to quantitatively analyze nuclear vs. cytoplasmic localization of other viral or cellular proteins without involving live imaging technology.

ICP0 undergoes nuclear-to-cytoplasmic translocation during HSV-1 infection. The molecular mechanism of this event is not known. Here we describe the use of confocal microscope as a tool to quantify ICP0 movement in HSV-1 infection, which lays the groundwork for quantitatively analyzing ICP0 translocation in future mechanistic studies.