The authors would like to thank Richard Rest for the generous gift of anthrolysin O, Michael Caparon for the generous gift of the SLO plasmid and Jonathon Franks for technical assistance. This work was funded by NIH grants T32CA82084 (PAK), and R01AI072083 (RDS).

1. Purification of Streptolysin O (SLO)
<ol>
	<li>Inoculate 20 ml overnight culture of BL21 GOLD cells containing pBADgIII-SLOhis plasmid9 into 0.5 L LB broth and add 500 &mu;l 50 mg/ml ampicillin. Shake culture at 225 rpm at 37 &deg; C until OD600 = 0.6, usually ~1.5 hr. Centrifuge 1 ml bacteria (considered T0) at 10,000 x g 5 min, dissolve pellet in 140 &mu;l 1x SDS sample buffer/1 OD and sonicate to shear DNA for protein purity analysis.</li>
	<li>Induce bacteria with the addition of...

<ol>
	<li>Walev, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delivery+of+proteins+into+living+cells+by+reversible+membrane+permeabilization+with+streptolysin-O.">Delivery of proteins into living cells by reversible membrane permeabilization with streptolysin-O.</a> Proc. Natl. Acad. Sci. U.S.A. 98, 3185-3190 (2001).</li><li>Walev, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potassium+regulates+IL-1+beta+processing+via+calcium-independent+phospholipase+A2.">Potassium regulates IL-1 beta processing via calcium-independent phospholipase A2.</a> J. Immunol. 164, 5120-5124 (2000).</li><li>Scolding, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vesicular+removal+by+oligodendrocytes+of+membrane+attack+complexes+formed+by+activated+complement.">Vesicular removal by oligodendrocytes of membrane attack complexes formed by activated complement.</a> Nature. 339, 620-622 (1989).</li><li>Keyel, 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=Streptolysin+O+clearance+through+sequestration+into+blebs+that+bud+passively+from+the+plasma+membrane.">Streptolysin O clearance through sequestration into blebs that bud passively from the plasma membrane.</a> J. Cell. Sci. 124, 2414-2423 (2011).</li><li>MacKenzie, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+secretion+of+interleukin-1beta+by+microvesicle+shedding.">Rapid secretion of interleukin-1beta by microvesicle shedding.</a> Immunity. 15, 825-835 (2001).</li><li>Qu, Y., Franchi, L., Nunez, G., Dubyak, 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=Nonclassical+IL-1+beta+secretion+stimulated+by+P2X7+receptors+is+dependent+on+inflammasome+activation+and+correlated+with+exosome+release+in+murine+macrophages.">Nonclassical IL-1 beta secretion stimulated by P2X7 receptors is dependent on inflammasome activation and correlated with exosome release in murine macrophages.</a> J. Immunol. 179, 1913-1925 (2007).</li><li>Andrei, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phospholipases+C+and+A2+control+lysosome-mediated+IL-1+beta+secretion:+Implications+for+inflammatory+processes.">Phospholipases C and A2 control lysosome-mediated IL-1 beta secretion: Implications for inflammatory processes.</a> Proc. Natl. Acad. Sci. U.S.A. 101, 9745-9750 (2004).</li><li>Franchi, L., Eigenbrod, T., Munoz-Planillo, R., Nunez, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+inflammasome:+a+caspase-1-activation+platform+that+regulates+immune+responses+and+disease+pathogenesis.">The inflammasome: a caspase-1-activation platform that regulates immune responses and disease pathogenesis.</a> Nat. Immunol. 10, 241-247 (2009).</li><li>Magassa, N., Chandrasekaran, S., Caparon, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Streptococcus+pyogenes+cytolysin-mediated+translocation+does+not+require+pore+formation+by+streptolysin+O.">Streptococcus pyogenes cytolysin-mediated translocation does not require pore formation by streptolysin O.</a> EMBO Rep. 11, 400-405 (2010).</li><li>Pinkney, M., Beachey, E., Kehoe, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+thiol-activated+toxin+streptolysin+O+does+not+require+a+thiol+group+for+cytolytic+activity.">The thiol-activated toxin streptolysin O does not require a thiol group for cytolytic activity.</a> Infect. Immun. 57, 2553-2558 (1989).</li><li>Watkins, S. C., Salter, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+connectivity+between+immune+cells+mediated+by+tunneling+nanotubules.">Functional connectivity between immune cells mediated by tunneling nanotubules.</a> Immunity. 23, 309-318 (2005).</li><li>McNeil, P. L., Vogel, S. S., Miyake, K., Terasaki, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patching+plasma+membrane+disruptions+with+cytoplasmic+membrane.">Patching plasma membrane disruptions with cytoplasmic membrane.</a> J. Cell. Sci. 113, 1891-1902 (2000).</li><li>Shannon, J. G., Ross, C. L., Koehler, T. M., Rest, R. F. <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+anthrolysin+O,+the+Bacillus+anthracis+cholesterol-dependent+cytolysin.">Characterization of anthrolysin O, the Bacillus anthracis cholesterol-dependent cytolysin.</a> Infect. Immun. 71, 3183-3189 (2003).</li></ol>

The techniques described here allow the examination of the responses of immune cells to bacterial toxins. The most critical step is the handling and dosing of the toxin. Toxin activity can be extremely variable, even between different aliquots of the same preparation, due to its fragility. This necessitates either testing each aliquot of toxin against a reference cell line or RBCs or using toxin gradients. Toxin gradients, as delivered by micropipette, allow the full spectrum of toxin-induced activities to be observed in...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Ni-NTA agarose</td>
 <td>Qiagen</td>
 <td>30210</td>
 <td></td>
 </tr>
 <tr>
 <td>polymixin-agarose</td>
 <td>Sigma</td>
 <td>P1411-5ML</td>
 <td></td>
 </tr>
 <tr>
 <td>Zeba Desalt Spin col</td>
 <td>Fisher</td>
 <td>PI-89891</td>
 <td></td>
 </tr>
 <tr>
 <td>sheep RBCs</td>
 <td>Fisher</td>
 <td>50-415-688</td>
 <td></td>
 </tr>
 <tr>
 <td>pBADgIII-SLO</td>
 <td>N/A</td>
 <td>N/A</td>
 <td>see ref9 </td>
 </tr>
 <tr>
 <td>Cy5 monoreactive dye</td>
 <td>GE Healthcare</td>
 <td>PA25001</td>
 <td></td>
 </tr>
 <tr>
 <td>Fura2-AM</td>
 <td>Life Technologies</td>
 <td>F1221</td>
 <td></td>
 </tr>
 <tr>
 <td>Calcein AM/ Ethidium homodimer</td>
 <td>Life Technologies</td>
 <td>L3224</td>
 <td></td>
 </tr>
 <tr>
 <td>Anti-CD11c-APC</td>
 <td>BD Biosciences</td>
 <td>550261</td>
 <td></td>
 </tr>
 <tr>
 <td>collagen-coated glass-bottom dish</td>
 <td>Mattek</td>
 <td>P35GCol-1.5-10-C</td>
 <td></td>
 </tr>
 <tr>
 <td>femto-tip II</td>
 <td>Fisher</td>
 <td>E5242957000</td>
 <td></td>
 </tr>
 <tr>
 <td>Microloader</td>
 <td>Fisher</td>
 <td>E5242956003</td>
 <td></td>
 </tr>
 <tr>
 <td>dextran Alexa 555</td>
 <td>Life Technologies</td>
 <td>D34679</td>
 <td></td>
 </tr>
 <tr>
 <td>Injectman NI 2</td>
 <td>Eppendorf</td>
 <td>920000029</td>
 <td></td>
 </tr>
 <tr>
 <td>FemtoJet</td>
 <td>Eppendorf</td>
 <td>5247 000.013</td>
 <td></td>
 </tr>
 <tr>
 	<td></td>
 <td></td>
 <td></td>
 <td>Table 1. List and source of specific reagents and equipment needed. Specific equipment and reagents used in this protocol, along with company and catalogue number are listed.</td>
 </tr>
 <tr>
 	<td></td>
 <td></td>
 <td></td>
 <td><table border="1">
 <tbody>
 <tr>
 <td>Buffer</td>
 <td>Composition</td>
 <td>Step Used</td>
 </tr>
 <tr>
 <td>Lyse/Wash</td>
 <td>50 mM NaH2PO4 300 mM NaCl 10 mM imidazole, pH 8.0</td>
 <td>1.4</td>
 </tr>
 <tr>
 <td>Tris/salt</td>
 <td>50 mM Tris, pH 8.0 300 mM NaCl</td>
 <td>1.7</td>
 </tr>
 <tr>
 <td>Elution buffer</td>
 <td>50 mM Tris, pH 8.0 300 mM NaCl 250 mM imidazole</td>
 <td>1.8</td>
 </tr>
 <tr>
 <td>RBC Assay buffer</td>
 <td>0.3% BSA 2 mM CaCl2 10 mM HEPES, pH 7.4</td>
 <td>2.1</td>
 </tr>
 <tr>
 <td>buffer R/B</td>
 <td>RPMI cell culture medium 2 mM CaCl2 0.5% BSA</td>
 <td>3.1</td>
 </tr>
 </tbody>
</table>
</td>
 </tr>
 <tr>
 	<td></td>
 <td></td>
 <td></td>
 <td>Table 2. List of buffers used in this protocol. The buffers used, their composition and the first step at which they are used in the protocol are listed.</td>
 </tr>
 </tbody>
</table>

visualization of bacterial toxin induced responses using live cell fluorescence microscopy

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external environment. The cell can either recover from this insult, which requires active membrane repair processes, or else die depending on the amount of toxin exposure and cell type1. In addition, these toxins induce strong inflammatory responses in infected hosts through activation of immune cells, including macrophages, which produce an array of pro-inflammatory cytokines2. Many Gram positive bacteria produce cholesterol binding toxins which have been shown to contribute to their virulence through largely uncharacterized mechanisms.
Morphologic changes in the plasma membrane of cells exposed to these toxins include their sequestration into cholesterol-enriched surface protrusions, which can be shed into the extracellular space, suggesting an intrinsic cellular defense mechanism3,4. This process occurs on all cells in the absence of metabolic activity, and can be visualized using EM after chemical fixation4. In immune cells such as macrophages that mediate inflammation in response to toxin exposure, induced membrane vesicles are suggested to contain cytokines of the IL-1 family and may be responsible both for shedding toxin and disseminating these pro-inflammatory cytokines5,6,7. A link between IL-1&beta; release and a specific type of cell death, termed pyroptosis has been suggested, as both are caspase-1 dependent processes8. To sort out the complexities of this macrophage response, which includes toxin binding, shedding of membrane vesicles, cytokine release, and potentially cell death, we have developed labeling techniques and fluorescence microscopy methods that allow for real time visualization of toxin-cell interactions, including measurements of dysfunction and death (Figure 1). Use of live cell imaging is necessary due to limitations in other techniques. Biochemical approaches cannot resolve effects occurring in individual cells, while flow cytometry does not offer high resolution, real-time visualization of individual cells. The methods described here can be applied to kinetic analysis of responses induced by other stimuli involving complex phenotypic changes in cells.

Typically 107-108 U/ml SLO can be obtained with a protein concentration of 4 mg/ml. The amount of toxin required for cell lysis varies by cell type, but is usually 125-500 U/ml SLO (Figure 2B). Cell types like macrophages can be more resistant (4000 U/ml) though others (especially T cell lines) are more sensitive. These sensitivities correspond with commercially available SLO. Toxin activity decreases roughly 2-fold with each freeze-thaw, so hemolytic assays with each batch or thaw ...

Watch this Scientific Journal Video about Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy at JoVE.com

Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

Methods for purifying the cholesterol binding toxin streptolysin O from recombinant E. coli and visualization of toxin binding to live eukaryotic cells are described. Localized delivery of toxin induces rapid and complex changes in targeted cells revealing novel aspects of toxin biology.

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external ...

visualization-bacterial-toxin-induced-responses-using-live-cell

Research

JoVE Journal

Immunology and Infection

11.7K visualizações.  University of Pittsburgh School of Medicine. Os métodos para a purificação do colesterol de ligação da toxina a partir de estreptolisina O recombinante E. coli E visualização de toxina se ligar a células eucarióticas vivo são descritos. Entrega localizada de toxina induz a mudanças rápidas e complexas em células-alvo, revelando novos aspectos da biologia toxina.