This work was funded by NIH grant R01 AI073783 to K. Teter. We thank Dr. Shane Massey for assistance in the development of the subcellular fractionation protocol and Helen Burress for critical reading of the manuscript.

1. Preparation of digitonin
<ol>
	<li>Add 500 &mu;L of 100% ethanol to a microcentrifuge tube and place it in a heat block set at 80 &deg;C for 10 min.</li>
	<li>Dissolve 2.5 mg of digitonin in 250 &mu;L of the heated ethanol to produce a 1% stock solution of digitonin.</li>
	<li>To generate a working solution of 0.04% digitonin, add 40 &mu;L of the digitonin stock solution to 960 &mu;L of HCN buffer (50 mM Hepes pH 7.5, 150 mM NaCl, 2 mM CaCl2, 10 mM N-ethylmaleimide, and a protease inhibitor cocktail).</li...

Comparison to existing methodology
Our SPR-based translocation assay represents a rapid, sensitive, and quantitative method to detect toxin delivery into the host cytosol. The technique does not require radiolabeling or other modifications to the toxin, and it can be applied to any toxin for which an anti-toxin A chain antibody is available. Existing methods to monitor toxin passage into the cytosol also rely upon a subcellular fractionation protocol to partition cell extracts into separate cyt...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>Digitonin</td>
 <td>Sigma</td>
 <td>D141</td>
 </tr>
 <tr>
 <td>Ethanol</td>
 <td>Acros</td>
 <td>61509-0010</td>
 </tr>
 <tr>
 <td>DMEM</td>
 <td>Invitrogen</td>
 <td>11995065</td>
 </tr>
 <tr>
 <td>Fetal Bovine Serum</td>
 <td>Atlanta Biologicals</td>
 <td>S11550</td>
 </tr>
 <tr>
 <td>Ganglioside GM1</td>
 <td>Sigma</td>
 <td>G7641</td>
 </tr>
 <tr>
 <td>CTA</td>
 <td>Sigma</td>
 <td>C2398</td>
 </tr>
 <tr>
 <td>PTS1</td>
 <td>List</td>
 <td>182</td>
 </tr>
 <tr>
 <td>NHS (N-Hydroxysuccinimide)</td>
 <td>Pierce</td>
 <td>24500</td>
 </tr>
 <tr>
 <td>EDC (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide)</td>
 <td>Thermo Scientific</td>
 <td>22981</td>
 </tr>
 <tr>
 <td>Ethanolamine </td>
 <td>Sigma</td>
 <td>E0135</td>
 </tr>
 <tr>
 <td>PBST</td>
 <td>Medicago</td>
 <td>09-8903-100</td>
 </tr>
 <tr>
 <td>Anti-CTA antibody</td>
 <td>Santa Cruz Biotech</td>
 <td>sc-80747</td>
 </tr>
 <tr>
 <td>Anti-CTB antibody</td>
 <td>Calbiochem</td>
 <td>227040</td>
 </tr>
 <tr>
 <td>Anti-PTS1 antibody</td>
 <td>Santa Cruz Biotech</td>
 <td>sc-57639 </td>
 </tr>
 <tr>
 <td>Refractometer</td>
 <td>Reichert</td>
 <td>SR7000, SR7000DC</td>
 </tr>
 <tr>
 <td>SPR sensor slides</td>
 <td>Reichert</td>
 <td>13206060</td>
 </tr>
 <tr>
 <td>Syringe pump</td>
 <td>Cole Palmer</td>
 <td>780200C</td>
 </tr>
 </tbody>
</table>

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

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.

Watch this Scientific Journal Video about Detection of Toxin Translocation into the Host Cytosol by Surface Plasmon Resonance at JoVE.com

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 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 ...

detection-toxin-translocation-into-host-cytosol-surface-plasmon

Research

JoVE Journal

Immunology and Infection

13.2K Aufrufe.  University of Central Florida. In diesem Bericht beschreiben wir, wie Oberflächen-Plasmon-Resonanz verwendet wird, um Toxin Einreise in den Aufnahmemitgliedstaat Zytosol zu erkennen. Dieser hochempfindliche Methode lassen sich quantitative Daten über die Höhe der zytosolischen Toxin geben, und es kann zu einer Reihe von Toxinen eingesetzt werden.