This study was supported by Public Health Service grant AI-34431 (to D.A.H.) from the National Institute of Allergy and Infectious Diseases and by VA Medical Research Funds (to D.A.H.).

1. Fixation of Leptospira to glass slides
<ol>
<li>Leptospira interrogans is a class BSL2 pathogen. Working with live cells it requires appropriate handling, such as wearing gloves, lab-coat and pipetting steps in a sterile-hood.</li>
<li>Grow Leptospira in EMJH medium11, supplemented with 1% rabbit serum at 30&deg; C until they reach mid- to late-log phase (density of 5 x 107 to 5 x 108 cells/ml) for approximately 6 days.</li>
<li>Harvest the culture by centrifugation at &tilde;2000 x g for 7 min at room temperature.</li> 
<li>Remove the supernatant by aspiration and gently resuspend the pellet in phosphate buffered saline (PBS)-5 mM MgCl2, pH7.2 to a final concentration of 5 x 108 cells/ml.</li>
<li>Add 1 ml of cell suspension to each well of two-well chamber glass slides and incubate at 30&deg; C for 80 min to allow cells to adhere.</li>
<li>Carefully remove the liquid containing unbound cells by aspiration.</li>
<li>Fix remaining intact bacteria to glass slides by adding 1 ml/well of 2% paraformaldehyde in PBS-5 mM MgCl2. Incubate for 40 min at 30&deg; C. These slides will be for assessment of surface-exposed proteins.</li>
<li>For control slides assessing sub-surface proteins, permeabilize the outer membrane by fixing with 1 ml/well of 100% ice-cold methanol. Incubate at -20&deg; C for 20 min. Methanol acts in several ways: it permeabilizes the outer membrane, denaturates the proteins, and fixes cells to glass slides.</li>
<li>Remove fixing agents by aspiration.</li>
</ol>
2. Labeling with specific antibodies
<ol>
<li>Block non-specific binding by adding 1 ml/well of blocking buffer (Difco Leptospira Enrichment EMJH). Incubate at 30&deg; C for 90 min.</li>
<li>Dilute the specific antibody (immune rabbit sera or mouse monoclonal antibodies, here polyclonal rabbit sera specific for OmpL5410, FlaA112, and OmpL113) and pre-immune rabbit sera or mouse ascetic fluid containing no antibody (when utilized as negative controls) in blocking buffer. Dilutions for each antibody have to be determined empirically depending on antibody titer, antigen-antibody reactivity and abundance of protein in the cell. The usual range is 1:50 to 1:600.</li>
<li>Remove the blocking buffer by aspiration and add 1 ml/well of diluted primary antibodies.</li>
<li>Incubate at 30&deg; C for 1h.</li>
<li>Remove the liquid by aspiration and wash the wells three times with PBS (1 ml/well).</li>
</ol>
3. Visualization of leptospires
<ol>
<li>Add 1 ml/well of Alexa Fluor 488-labeled secondary antibodies (either goat anti-rabbit IgG or goat anti-mouse IgG) diluted 1:2000 and fluorescent nucleic acid stain, 4&#x2019;6-diamidino-2-phenyl-indole dihydrochloride (DAPI) diluted to a final concentration of 0.25 &mu;g/ml in blocking buffer. This step ensures detection of antibody binding and the presence of all spirochetes independent of antibody binding, respectively.</li>
<li>Incubate the slides at 30&deg; C for 45 min.</li>
<li>Remove the liquid by aspiration and wash the wells twice with PBS and once with distilled water (1 ml/well).</li>
<li>Remove the chambers and the adhesive strip from the glass slides and air-dry for &tilde;10 min.</li>
<li>Add ProLong Gold anti-fade mounting medium (2 x 20 &mu;l per slide) and a 24 x 50 mm cover slip.</li>
<li>Incubate overnight at room temperature in the dark. This step is necessary to allow the mounting medium to cure (harden).</li>
<li>Seal the cover slip with nail polish.</li>
<li>Visualize the staining by fluorescence microscopy using a cyan/blue detection filter for DAPI and a green detection filter for Alexa Fluor 488. To ensure that an accurate representation of the samples is made, make sure you evaluate the entire chamber area for each sample.</li>
<li>Record the data by imaging a representative field for each sample. When imaging Alexa Fluor 488 fluorescence, use the same exposure time for all samples. Using a consistent exposure time will allow more accurate comparison of results with test antigens versus controls.</li>
</ol>
4. Representative Results: 
Examples of surface IFA results with Leptospira interrogans cells using antibodies against surface and sub-surface proteins are shown in Figure 2. A test is considered positive when substantial fluorescence is detected in samples with intact leptospires for a protein of interest that is surface exposed (Fig. 2A). Usually, in a positive test (here, surface-exposed OmpL54), approximately same fluorescence intensity is observed in both intact and permeabilized cells (Fig. 2A) It is essential to include a negative control for outer-membrane integrity using antibodies against a subsurface, preferably periplasmic protein (e.g. leptospiral FlaA1, Fig. 2B). Only when the data show that the outer membrane has remained intact during the experiment, i.e. antibodies against a sub-surface protein do not react to intact cells (no fluorescence or much weaker fluorescence when compared to permeabilized sample as in Fig. 2B), can it be concluded that the protein(s) of interest is surface exposed. Inclusion of experiments with pre-immune sera (in the case of polyclonal antibodies) as an additional negative control is essential for unambiguous data and should not yield substantial fluorescence in intact or permeabilized cells (Fig. 2A, 2C). DAPI stain is necessary to visualize leptospires when negative results (no Alexa Fluor 488 fluorescence) are observed. Counterstaining with propidium iodide is an acceptable alternative to DAPI.
This method is qualitative rather than quantitative as brighter fluorescence may be due to multiple surface exposed antigenic epitopes being recognized in a single antigen relative to other antigens of equal abundance but with fewer surface exposed epitopes. Therefore the fluorescence intensities can only be used to compare reactivity of the same antibodies, such as intact versus permeabilized cells and not fluorescence intensities between different proteins. This distinction is important when an ambiguous result is obtained where significantly less fluorescence is observed in intact cells than in permeabilized cells (Fig. 2C). Such a result indicates that either the protein is in a sub-surface location (with some non-specific background staining of intact cells), the antibodies are binding preferentially to non-native epitopes that are exposed after methanol permeabilization or a protein might have multiple localization sites as described for Erp and Elp proteins of another spirochete, Borrelia burgdorferi14. In either case, an ambiguous IFA result such as this requires alternative approaches such as surface biotinylation or surface proteolysis to determine whether the protein of interest is surface-exposed or not 10. 
<img src="/files/ftp_upload/2805/2805fig1.jpg" alt="Figure 1"/> 
Figure 1. Flow chart for the surface immuno-fluorescence assay of Leptospira interrogans spirochetes. First, 1 ml of cell suspension is added to each well of two-well chamber slides (at least two slides for each experiment) and incubated for leptospires to adhere. Next, leptospires are fixed to chamber slides by adding 1 ml/well of 2% paraformaldehyde (PFA) for samples with intact outer membrane (OM) and 1 ml/well of 100% cold methanol for samples with permeabilized OM. Next, leptospires are incubated with antibodies recognizing surface-exposed proteins along with control antibodies for subsurface proteins (Primary Ab&#x2019;s) at 30&deg; C for 90 min. Then, primary antibodies are detected by incubation with 1 ml/well of Alexa Fluor 488 conjugated secondary antibodies. Finally, cells are visualized by fluorescence microscopy.
<img src="/files/ftp_upload/2805/2805fig2.jpg" alt="Figure 2"/> 
Figure 2. Surface immunofluorescence analysis of leptospiral proteins. Leptospira interrogans spirochetes were probed with immune and pre-immune sera, and negative results were paired with a DAPI counterstain to demonstrate the presence of spirochetes. A green fluorescence filter was used to detect Alexa Fluor 488 labeled secondary antibody binding to surface exposed proteins and blue/cyan fluorescence filter to detect DAPI binding to cell DNA. A) Leptospires probed with antibodies recognizing a surface exposed protein, OmpL5410. B) Cells probed with antibodies against a periplasmic subsurface protein, FlaA1 (negative control). C) Cells probed with antibodies against leptospiral porin, OmpL1. Binding of rabbit sera to leptospires were detected with Alexa Fluor 488 conjugated goat anti-rabbit IgG fragments. All images are taken after 4 sec long exposure.

<ol>
	<li>Achouak, W., Heulin, T., Pages, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+facets+of+bacterial+porins.">Multiple facets of bacterial porins.</a> FEMS Microbiol Lett. 199, 1-7 (2001).</li><li>Haake, D. A., Matsunaga, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+leptospiral+outer+membrane+and+description+of+three+novel+leptospiral+membrane+proteins.">Characterization of the leptospiral outer membrane and description of three novel leptospiral membrane proteins.</a> Infect Immun. 70, 4936-4945 (2002).</li><li>Haake, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+the+surface+of+Leptospira+interrogans+serovar+grippotyphosa+during+in+vitro+cultivation.">Changes in the surface of Leptospira interrogans serovar grippotyphosa during in vitro cultivation.</a> Infect Immun. 59, 1131-1140 (1991).</li><li>Nally, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+proteomic+analysis+of+outer+membrane+vesicles+from+a+clinical+isolate+of+Leptospira+interrogans+serovar+Copenhageni.">Purification and proteomic analysis of outer membrane vesicles from a clinical isolate of Leptospira interrogans serovar Copenhageni.</a> Proteomics. 5, 144-152 (2005).</li><li>Zuerner, R. L., Knudtson, W., Bolin, C. A., Trueba, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+outer+membrane+and+secreted+proteins+of+Leptospira+interrogans+serovar+pomona.">Characterization of outer membrane and secreted proteins of Leptospira interrogans serovar pomona.</a> Microb Pathog. 10, 311-322 (1991).</li><li>Cullen, P. A., Haake, D. A., Adler, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Outer+membrane+proteins+of+pathogenic+spirochetes.">Outer membrane proteins of pathogenic spirochetes.</a> FEMS Microbiol Rev. 28, 291-318 (2004).</li><li>Haake, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spirochaetal+lipoproteins+and+pathogenesis.">Spirochaetal lipoproteins and pathogenesis.</a> Microbiology. 146, 1491-1504 (2000).</li><li>Koebnik, R., Locher, K. P., Gelder, P. V. a. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+function+of+bacterial+outer+membrane+proteins:+barrels+in+a+nutshell.">Structure and function of bacterial outer membrane proteins: barrels in a nutshell.</a> Mol Microbiol. 37, 239-253 (2000).</li><li>Schulz, G., Benz, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The structures of general porins. , (2004).</li><li>Pinne, M., Haake, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comprehensive+approach+to+identification+of+surface-exposed,+outer+membrane-spanning+proteins+of+Leptospira+interrogans.">A comprehensive approach to identification of surface-exposed, outer membrane-spanning proteins of Leptospira interrogans.</a> PLoS One. 4, e6071-e6071 (2009).</li><li>Johnson, R. C., Rogers, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolism+of+leptospires.+II.+The+action+of+8-azaguanine.">Metabolism of leptospires. II. The action of 8-azaguanine.</a> Can J Microbiol. 13, 1621-1629 (1967).</li><li>Cullen, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surfaceome+of+Leptospira+spp.">Surfaceome of Leptospira spp.</a> Infect Immun. 73, 4853-4863 (2005).</li><li>Haake, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+cloning+and+sequence+analysis+of+the+gene+encoding+OmpL1,+a+transmembrane+outer+membrane+protein+of+pathogenic+Leptospira+spp.">Molecular cloning and sequence analysis of the gene encoding OmpL1, a transmembrane outer membrane protein of pathogenic Leptospira spp.</a> J Bacteriol. 175, 4225-4234 (1993).</li><li>Hefty, P. S., Jolliff, S. E., Caimano, M. J., Wikel, S. K., Akins, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+temporal+and+spatial+patterns+of+outer+surface+lipoprotein+expression+generate+population+heterogeneity+and+antigenic+diversity+in+the+Lyme+disease+spirochete,+Borrelia+burgdorferi.">Changes in temporal and spatial patterns of outer surface lipoprotein expression generate population heterogeneity and antigenic diversity in the Lyme disease spirochete, Borrelia burgdorferi.</a> Infect Immun. 70, 3468-3478 (2002).</li><li>Noppa, L., &#214;stberg, Y., Lavrinovicha, M., Bergstr&#246;m, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=P13+an+integral+membrane+protein+of+Borrelia+burgdorferi,+is+C-terminally+processed+and+contains+surface-exposed+domains.">P13 an integral membrane protein of Borrelia burgdorferi, is C-terminally processed and contains surface-exposed domains.</a> Infect Immun. 69, 3323-3334 (2001).</li><li>Parveen, N., Leong, J. M. <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+a+candidate+glycosaminoglycan-binding+adhesin+of+the+Lyme+disease+spirochete+Borrelia+burgdorferi.">Identification of a candidate glycosaminoglycan-binding adhesin of the Lyme disease spirochete Borrelia burgdorferi.</a> Mol Microbiol. 35, 1220-1234 (2000).</li><li>Ristow, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+OmpA-like+protein+Loa22+is+essential+for+leptospiral+virulence.">The OmpA-like protein Loa22 is essential for leptospiral virulence.</a> PLoS Pathog. 3, e97-e97 (2007).</li></ol>

No conflicts of interest declared.

Our surface IFA technique is similar to that used to demonstrate the surface exposure of various borrelial 15, 16 and leptospiral 12, 17 proteins. The details of the surface IFA method described here are designed to minimize the manipulation of cells in an effort to maintain outer membrane integrity while taking advantage of the tendency of Leptospira cells to adhere to glass slides. The method involves a lower concentration (2% versus 4%) of paraformaldehyde, washing with outer-membrane stabilizin...

<table border="1">
	<tbody>
 <tr>
 	<th>Name of the reagent</th>
 <th>Company</th>
 <th>Catalogue number</th>
 <th>Comments (optional)</th>
 </tr>
 <tr>
 	<td>Two-well chamber glass slides</td>
 <td>Lab-Tek</td>
 <td>177380

</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Rabbit serum</td>
 <td>Rockland Immunochemicals</td>
 <td>D209-00-0100</td>
 <td>&nbsp;</td>
 </tr>

 <tr>
 	<td>Leptospira Enrichment EMJH</td>
 <td>BD Difco</td>
 <td>279510</td>
 <td>&nbsp;</td>
 </tr>

 <tr>
 	<td>Alexa Fluor 488 goat anti-rabbit IgG (H+L)</td>
 <td>Invitrogen/Molecular Probes</td>
 <td>A-11034</td>
 <td>&nbsp;</td>
 </tr>

 <tr>
 	<td>Alexa Fluor 488 goat anti-mouse IgG (H+L)</td>
 <td>Invitrogen/Molecular Probes</td>
 <td>A-11029</td>
 <td>&nbsp;</td>
 </tr>

 <tr>
 	<td>4&#x2019;6-diamidino-2-phenyl-indole dihydrochloride (DAPI)</td>
 <td>Invitrogen/Molecular Probes</td>
 <td>D1306</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>ProLong Gold anti-fade mounting medium</td>
 <td>Invitrogen/Molecular Probes</td>
 <td>P36930</td>
 <td>Equilibrate to room temperature before use</td>
 </tr>
 <tr>
 	<td>Fisherfinest Premium Cover Glass</td>
 <td>Fisher</td>
 <td>12-544-14</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Fluorescence microscope</td>
 <td>Zeiss</td>
 <td>Axioskop 40</td>
 <td>&nbsp;</td>
 </tr>
 

 </tbody>
</table>

immuno-fluorescence assay of leptospiral surface-exposed proteins

Bacterial surface proteins are involved in direct contact with host cells and in uptake of nutrients from the environment 1. For this reason, cellular localization can provide insights into the functional role of bacterial proteins. Surface localization of bacterial proteins is a key step towards identification of virulence factors involved in mechanisms of pathogenicity. 
Methods for fractionating leptospiral membranes 2-5 may be selective for a certain class of outer-membrane proteins (OMPs), such as lipoproteins vs. transmembrane OMPs, and therefore lead to misclassification. This likely is due to structural differences and how they are associated to the outer membrane. Lipoproteins are associated with membranes via a hydrophobic interaction between the N-terminal lipid moiety (three fatty acids) and the lipid bilayer phospholipids 6, 7. In contrast, transmembrane OMPs are typically integrated into the lipid bilayer by amphipathic &beta;-sheets arranged in a barrel-like structure 8, 9. In addition, presence of a protein in the outer-membrane does not necessarily guarantee that the protein or its domains are exposed on the surface. Spirochetal outer membranes are known to be fragile and therefore necessitate methods involving gentle manipulation of cells and inclusion of sub-surface protein controls to assess the integrity of the outer membrane.
Here, we present an immunofluorescence assay (IFA) method to directly assess surface exposure of proteins on intact leptospires. This method is based on recognition of leptospiral surface proteins by antigen-specific antibodies. Herein, antibodies specific for OmpL5410 are detetcted aftero binding to native, surface exposed epitopes. Comparison of antibody reactivity to intact versus permeabilized cells enables evaluation of cellular distribution and whether or not a protein is selectively present on leptospiral surface. The integrity of outer membrane should be assessed using antibody to one or more subsurface proteins, preferably located in the periplasm.
The surface IFA method can be used to analyze surface exposure of any leptospiral protein to which specific antibodies are available. Both the usefulness and limitation of the method depends on whether the antibodies employed are able to bind to native epitopes. Since antibodies often are raised against recombinant proteins, epitopes of native, surface-exposed proteins may not be recognized. Nevertheless, the surface IFA method is a valuable tool for studying components of intact bacterial surfaces. This method can be applied not only for leptospires but also other spirochetes and gram-negative bacteria. For stronger conclusions regarding surface-exposure of OMPs, a comprehensive approach involving several cell localization methods is recommended 10.

Watch this Scientific Journal Video about Immuno-fluorescence Assay of Leptospiral Surface-exposed Proteins at JoVE.com

Immuno-fluorescence Assay of Leptospiral Surface-exposed Proteins

An efficient method to assess surface-exposure of leptospiral proteins is described. The method is specifically designed to avoid disruption of the fragile outer membrane of leptospiral cells. This technique requires employment of several negative controls to assess the integrity of the outer membrane and specificity of antibody reaction.

Bacterial surface proteins are involved in direct contact with host cells and in uptake of nutrients from the environment 1. For this reason, cellular ...

immuno-fluorescence-assay-of-leptospiral-surface-exposed-proteins

Research

JoVE Journal

Immunology and Infection

15.7K Views.  University of California, Los Angeles. An efficient method to assess surface-exposure of leptospiral proteins is described. The method is specifically designed to avoid disruption of the fragile outer membrane of leptospiral cells. This technique requires employment of several negative controls to assess the integrity of the outer membrane and specificity of antibody reaction.

Video: Immuno-fluorescence Assay of Leptospiral Surface-exposed Proteins

A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex (pMHC) proteins expressed on the surface of antigen presenting cells (APCs). The TCR complex is composed of the ligand binding TCR&alpha;&beta; heterodimer that associates non-covalently with CD3 dimers (the &#949;&delta; and &#949;&gamma; heterodimers and the &zeta;&zeta; homodimer)1. Upon engagement of the receptor, the CD3 &zeta; chains are phosphorylated by the Src family kinase, Lck. This leads to the recruitment of the Syk family kinase, Zap70, which is then phosphorylated and activated by Lck. After that, Zap70 phosphorylates the adapter proteins LAT and SLP76, initiating the formation of the proximal signaling complex containing a large number of different signaling molecules2. 
The formation of this complex eventually results in calcium and Ras-dependent transcription factor activation and the consequent initiation of a complex series of gene expression programs that give rise to T cell differentiation2. TCR signals (and the resulting state of differentiation) are modulated by many other factors, including antigen potency and crosstalk with co-stimulatory/co-inhibitory, chemokine, and cytokine receptors 3-4. Studying the spatial and temporal organization of the proximal signaling complex under various stimulation conditions is, therefore, key to understanding the TCR signaling pathway as well as its regulation by other signaling pathways.
One very useful model system to study signaling initiated by the TCR at the plasma membrane in T cells is glass-supported lipid bilayers, as described previously5-6. They can be utilized to present antigenic pMHC complexes, adhesion, and co-stimulatory molecules to T cells-serving as artificial APCs. By imaging the T cells interacting with the lipid bilayer using total internal reflection fluorescence microscopy (TIRFM), we can restrict the excitation to within 100 nm of the space between the glass and the cell surface 7-8. This allows us to image primarily the signaling events occurring at the plasma membrane. As we are interested in imaging the recruitment of signaling proteins to the TCR complex, we describe a two-camera TIRF imaging system wherein the TCR, labeled with fluorescent Fab (fragment antigen binding) fragments of the H57 antibody (purified from hybridoma H57-597, ATCC, ATCC Number:HB-218) which is specific for TCR&beta;, and signaling proteins, tagged with GFP, may be imaged simultaneously and in real time. This strategy is necessary due to the highly dynamic nature of both the T cells and of the signaling events that are occurring at the TCR. This imaging modality has allowed researchers to image single ligands 9-11 as well as recruitment of signaling molecules to activated receptors and is an excellent system to study biochemistry in-situ12-16.

The compartmentalization of proteins either within the plasma membrane or into intracellular locations is one regulatory mechanism that can greatly influence signaling outcomes; hence, to understand signaling it is important to study the spatial and temporal behavior of the proteins involved. We describe here a TIRF microscopy based system to study signal transduction in T cells, but is broadly applicable.

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex ...

Plaque Assay for Murine Norovirus

Murine norovirus (MNV) is the only member of the Norovirus genus that efficiently grows in tissue culture 1, 2. Cell lysis and cytopathic effect (CPE) are observed during MNV-1 infection of murine dendritic cells or macrophages 1. This property of MNV-1 can be used to quantify the number of infectious particles in a given sample by performing a plaque assay 1. The plaque assay relies on the ability of MNV-1 to lyse cells and to form holes in a confluent cell monolayer, which are called plaques 3.
Multiple techniques can be used to detect viral infections in tissue culture, harvested tissue, clinical, and environmental samples, but not all measure the number of infectious particles (e.g. qRT-PCR). One way to quantify infectious viral particles is to perform a plaque assay 3, which will be described in detail below. A variation on the MNV plaque assay is the fluorescent focus assay, where MNV antigen is immunostained in cell monolayers 4. This assay can be faster, since viral antigen expression precedes plaque formation. It is also useful for titrating viruses unable to form plaques. However, the fluorescent focus assay requires additional resources beyond those of the plaque assay, such as antibodies and a microscope to count focus-forming units. Infectious MNV can also be quantified by determining the 50% Tissue Culture Infective Dose (TCID50) 3. This assay measures the amount of virus required to produce CPE in 50% of inoculated tissue culture cells by endpoint titration 5. However, its limit of detection is higher compared to a plaque assay 4.
In this article, we describe a plaque assay protocol that can be used to effectively determine the number of infectious MNV particles present in biological or environmental samples 1, 4, 6. This method is based on the preparation of 10-fold serial dilutions of MNV-containing samples, which are used to inoculate a monolayer of permissive cells (RAW 264.7 murine macrophage cells). Virus is allowed to attach to the cell monolayer for a given period of time and then aspirated before covering cells with a mixture of agarose and cell culture media. The agar enables the spread of viral progeny to neighboring cells while limiting spread to distantly located cells. Consequently, infected cells are lysed and form holes in the monolayer known as plaques. Upon sufficient spread of virus, plaques become visible following staining of cells with dyes, like neutral red, methylene blue, or crystal violet. At low dilutions, each plaque originates from one infectious viral particle and its progeny, which spread to neighboring cells. Thus, counting the number of plaques allows one to calculate plaque-forming units (PFU) present in the undiluted sample 3.

Here we describe a method to quantify infectious particles of murine norovirus (MNV), which is the only norovirus that efficiently replicates in cell culture. The plaque assay takes advantage of MNV&#x2019;s tropism for murine macrophages and can be adapted for use with biological or environmental samples containing MNV.

Murine norovirus (MNV) is the only member of the Norovirus genus that efficiently grows in tissue culture 1, 2. Cell lysis and cytopathic effect (CPE) are ...

Methodology for the Efficient Generation of Fluorescently Tagged Vaccinia Virus Proteins

Tagging of viral proteins with fluorescent proteins has proven an indispensable approach to furthering our understanding of virus-host interactions. Vaccinia virus (VACV), the live vaccine used in the eradication of smallpox, is particularly amenable to fluorescent live-cell microscopy owing to its large virion size and the ease with which it can be engineered at the genome level. We report here an optimized protocol for generating recombinant viruses. The minimal requirements for targeted homologous recombination during vaccinia replication were determined, which allows the simplification of construct generation. This enabled the alliance of transient dominant selection (TDS) with a fluorescent reporter and metabolic selection to provide a rapid and modular approach to fluorescently label viral proteins. By streamlining the generation of fluorescent recombinant viruses, we are able to facilitate downstream applications such as advanced imaging analysis of many aspects of the virus-host interplay that occurs during virus replication.

A rapid and modular protocol for the generation of recombinant vaccinia viruses expressing fluorescently&#160;tagged proteins simultaneously using the method of transient dominant selection is described here.

Tagging of viral proteins with fluorescent  proteins has proven an indispensable approach to furthering our understanding  of virus-host interactions. ...

Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in cattle1. The parasite alternates between the bloodstream of the mammalian host and the tsetse fly vector. The composition of many cellular organelles changes in response to these different extracellular conditions2-5.
Glycosomes are highly specialized peroxisomes in which many of the enzymes involved in glycolysis are compartmentalized. Glycosome composition changes in a developmental and environmentally regulated manner4-11. Currently, the most common techniques used to study glycosome dynamics are electron and fluorescence microscopy; techniques that are expensive, time and labor intensive, and not easily adapted to high throughput analyses.
To overcome these limitations, a fluorescent-glycosome reporter system in which enhanced yellow fluorescent protein (eYFP) is fused to a peroxisome targeting sequence (PTS2), which directs the fusion protein to glycosomes12, has been established. Upon import of the PTS2eYFP fusion protein, glycosomes become fluorescent. Organelle degradation and recycling results in the loss of fluorescence that can be measured by flow cytometry. Large numbers of cells (5,000 cells/sec) can be analyzed in real-time without extensive sample preparation such as fixation and mounting. This method offers a rapid way of detecting changes in organelle composition in response to fluctuating environmental conditions.

Glycosome dynamics in African trypanosomes are difficult to study by traditional cell biology techniques such as electron and fluorescence microscopy. As a means of observing dynamic organelle behavior, a fluorescent-organelle reporter system has been used in conjunction with flow cytometry to monitor real-time glycosome dynamics in live parasites.

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in ...

Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysis by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.

Here, we present phenomic approaches for the functional characterization of putative phage genes. Techniques include a developed assay capable of monitoring host anabolic metabolism, the Multi-phenotype Assay Plates (MAPs), in addition to the established method of metabolomics, capable of measuring effects to catabolic metabolism.

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage ...

Highly Sensitive Assay for Measurement of Arenavirus-cell Attachment

Arenaviruses are a family of enveloped RNA viruses that cause severe human disease. The first step in the arenavirus life cycle is attachment of viral particles to host cells. While virus-cell attachment can be measured through the use of virions labeled with biotin, radioactive isotopes, or fluorescent dyes, these approaches typically require high multiplicities of infection (MOI) to enable detection of bound virus. We describe a quantitative (q)RT-PCR-based assay that measures Junin virus strain Candid 1 attachment via quantitation of virion-packaged viral genomic RNA. This assay has several advantages including its extreme sensitivity and ability to measure attachment over a large dynamic range of MOIs without the need to purify or label input virus. Importantly, this approach can be easily tailored for use with other viruses through the use of virus-specific qRT-PCR reagents. Further, this assay can be modified to permit measurement of particle endocytosis and genome uncoating. In conclusion, we describe a simple, yet robust assay for highly sensitive measurement of arenavirus-cell attachment.

The first step in the arenavirus life cycle is attachment of viral particles to host cells. We report a quantitative (q)RT-PCR-based assay for ultrasensitive detection and quantitation of arenavirus attachment events.

Arenaviruses are a family of enveloped RNA viruses that cause severe human disease. The first step in the arenavirus life cycle is attachment of viral ...

Optimization of a Quantitative Micro-neutralization Assay

The micro-neutralization (MN) assay is a standard technique for measuring the infectivity of the influenza virus and the inhibition of virus replication. In this study, we present the protocol of an imaging-based MN assay to quantify the true antigenic relationships between viruses. Unlike typical plaque reduction assays that rely on visible plaques, this assay quantitates the entire infected cell population of each well. The protocol matches the virus type or subtype with the selection of cell lines to achieve maximum infectivity, which enhances sample contrast during imaging and image processing. The introduction of quantitative titration defines the amount of input viruses of neutralization and enables the results from different experiments to be comparable. The imaging setup with a flatbed scanner and free downloadable software makes the approach high throughput, cost effective, user friendly, and easy to deploy in most laboratories. Our study demonstrates that the improved MN assay works well with the current circulating influenza A(H1N1)pdm09, A(H3N2), and B viruses, without being significantly influenced by amino acid substitutions in the neuraminidase (NA) of A(H3N2) viruses. It is particularly useful for the characterization of viruses that either grow to low HA titer and/or undergo an abortive infection resulting in an inability to form plaques in cultured cells.

This study describes an imaging-based micro-neutralization assay to analyze the antigenic relationships between viruses. The protocol employs a flatbed scanner and has four steps, including titration, titration quantitation, neutralization, and neutralization quantitation. The assay works well with current circulating influenza A(H1N1)pdm09, A(H3N2), and B viruses.

The micro-neutralization (MN) assay is a standard technique for measuring the infectivity of the influenza virus and the inhibition of virus replication. ...

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. Whereas Pd transport of small molecules, such as ions and water, is presumed to occur passively, cell-to-cell transport of biological macromolecules, such proteins, most likely occurs via an active mechanism that involves specific targeting signals on the transported molecule. The scarcity of identified plasmodesmata (Pd) localization signals (PLSs) has severely restricted the understanding of protein-sorting pathways involved in plant cell-to-cell macromolecular transport and communication. From a wealth of plant endogenous and viral proteins known to traffic through Pd, only three PLSs have been reported to date, all of them from endogenous plant proteins. Thus, it is important to develop a reliable and systematic experimental strategy to identify a functional PLS sequence, that is both necessary and sufficient for Pd targeting, directly in the living plant cells. Here, we describe one such strategy using as a paradigm the cell-to-cell movement protein (MP) of the Tobacco mosaic virus (TMV). These experiments, that identified and characterized the first plant viral PLS, can be adapted for discovery of PLS sequences in most Pd-targeted proteins.

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Plant intercellular connections, the plasmodesmata (Pd), play central roles in plant physiology and plant-virus interactions. Critical to Pd transport are sorting signals that direct proteins to Pd. However, our knowledge about these sequences is still in its infancy. We describe a strategy to identify Pd localization signals in Pd-targeted proteins.

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. ...

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident STIM1 regulates the activity of PM Orai1 channels in a process known as store operated calcium (Ca2+) entry which is the principal Ca2+ signaling process that drives the immune response. STIM1 undergoes post-translational N-glycosylation at two luminal Asn sites within the Ca2+ sensing domain of the molecule. However, the biochemical, biophysical, and structure biological effects of N-glycosylated STIM1 were poorly understood until recently due to an inability to readily obtain high levels of homogeneous N-glycosylated protein. Here, we describe the implementation of an in vitro chemical approach which attaches glucose moieties to specific protein sites applicable to understanding the underlying effects of N-glycosylation on protein structure and mechanism. Using solution nuclear magnetic resonance spectroscopy we assess both efficiency of the modification as well as the structural consequences of the glucose attachment with a single sample. This approach can readily be adapted to study the myriad glycosylated proteins found in nature.

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Biochemical and structural analyses of glycosylated proteins require relatively large amounts of homogeneous samples. Here, we present an efficient chemical method for site-specific glycosylation of recombinant proteins purified from bacteria by targeting reactive Cys thiols.

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident ...

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and cellular proteins by mass spectrometry. Although proteins that interact with viral genomes play major roles in determining the outcome of infection, a comprehensive analysis of viral genome associated proteins was not previously feasible. Here we demonstrate a method that enables the direct purification of HSV-1 genomes from infected cells. Replicating viral DNA is selectively labeled with modified nucleotides that contain an alkyne functional group. Labeled DNA is then specifically and irreversibly tagged via the covalent attachment of biotin azide via a copper(I)-catalyzed azide-alkyne cycloaddition or click reaction. Biotin-tagged DNA is purified on streptavidin-coated beads and associated proteins are eluted and identified by mass spectrometry. This method enables the selective targeting and isolation of HSV-1 replication forks or whole genomes from complex biological environments. Furthermore, adaptation of this approach will allow for the investigation of various aspects of herpesviral infection, as well as the examination of the genomes of other DNA viruses.

The goal of this protocol is to specifically tag and selectively isolate viral DNA from infected cells for the characterization of viral genome associated proteins.