Parts of this study were kindly supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 1027, A6).

NOTE: An overview of the general experimental workflow is depicted in Figure 1.
Caution: RTA is highly toxic for humans. Safety lab permission S2 (biosafety level 2 equivalent) is needed. Please wear gloves during the entire experiment.
1. Heterologous Expression of His-tagged RTA in Escherichia coli
<ol>
	<li>Transform E. coli cells with the expression plasmid pET24-RTA(His)6 or the empty vector pET24a<su...

<ol>
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S., Yen, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+and+functional+properties+of+genetically+engineered+ricin+B+chain+lacking+galactose-binding+activity.">Expression and functional properties of genetically engineered ricin B chain lacking galactose-binding activity.</a> Biochim Biophys Acta. 1049 (2), 151-157 (1990).</li><li>Wales, R., Roberts, L. M., Lord, 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=Addition+of+an+endoplasmic+reticulum+retrieval+sequence+to+ricin+A+chain+significantly+increases+its+cytotoxicity+to+mammalian+cells.">Addition of an endoplasmic reticulum retrieval sequence to ricin A chain significantly increases its cytotoxicity to mammalian cells.</a> J Biol Chem. 268 (32), 23986-23990 (1993).</li><li>Breslow, D. 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=A+comprehensive+strategy+enabling+high-resolution+functional+analysis+of+the+yeast+genome.">A comprehensive strategy enabling high-resolution functional analysis of the yeast genome.</a> Nat Methods. 5 (8), 711-718 (2008).</li><li>Jablonowski, D., Schaffrath, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zymocin,+a+composite+chitinase+and+tRNase+killer+toxin+from+yeast.">Zymocin, a composite chitinase and tRNase killer toxin from yeast.</a> Biochem Soc Trans. 35 (Pt 6), 1533-1537 (2007).</li><li>Jablonowski, D., Schaffrath, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Saccharomyces+cerevisiae+RNA+polymerase+II+is+affected+by+Kluyveromyces+lactis+zymocin.">Saccharomyces cerevisiae RNA polymerase II is affected by Kluyveromyces lactis zymocin.</a> J Biol Chem. 277 (29), 26276-26280 (2002).</li><li>Jablonowski, D., Frohloff, F., Fichtner, L., Stark, M. J., Schaffrath, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kluyveromyces+lactis+zymocin+mode+of+action+is+linked+to+RNA+polymerase+II+function+via+Elongator.">Kluyveromyces lactis zymocin mode of action is linked to RNA polymerase II function via Elongator.</a> Mol Microbiol. 42 (4), 1095-1105 (2001).</li></ol>

The authors have nothing to disclose.

When performing the above protocol, we recommend the following suggestions to achieve a successful outcome of the experiment.
For heterologous protein expression, it is important to not exceed the IPTG concentration of 1 mM. IPTG concentrations &#62;1 mM inhibit promoter-induced RTA expression and lead to lower toxin yields. Furthermore, cells should not be cultivated at temperatures higher than 28 &#176;C to prevent inclusion body formation, inefficient folding, and toxin inactivation. RTA ex...

Patients suffering from infections by toxin producing bacteria represent a severe medical and financial burden for each social health care system, in particular since efficient therapeutic treatments are still largely missing. To develop new therapeutic strategies, the complex intoxication mechanisms of medically relevant A/B toxins such as cholera toxin, Shiga toxin, or ricin need to be fully understood at the molecular level based on novel powerful assays that have to be implemented.
In recent years, several studies attempted to analyze A/B toxin transport in yeast and mammalian cells by using time-consuming and cost-intensive methods such as radioactive toxin labeling1,2 as well as siRNA-based screening approaches3. In some cases, toxin trafficking has been visualized in vivo by fluorescence microscopy after chemical and/or genetic coupling of individual toxin subunits with fluorophores, quantum dots, or fluorescent proteins4,5. Unfortunately, such modifications often lead to inactive and/or altered natural properties of the toxins. Another elegant way to indirectly answer a wide variety of scientific questions is the use of reporter systems based on enzymes such as lacZ, luciferase, or fluorescent proteins (e.g. GFP or Discosoma sp. red fluorescent protein (dsRed)).
In this manuscript, a simple protocol is described which identifies cellular components required for the intracellular transport of extracellularly applied RTA in S. cerevisiae. Thereby, a fluorescence-reporter plasmid containing an N-terminal ER-import signal followed by GFP acts as a protein biosynthesis sensor, which indirectly measures RTA-mediated protein translation inhibition by GFP fluorescence emission after in vivo translation6. In case that RTA endocytosis and/or intracellular trafficking is negatively (or positively) affected in a particular yeast deletion mutant compared to wild-type, this can be detected through an increase (or decrease) in GFP fluorescence emission6.
So far, all methods analyzing RTA transport in yeast cells were restricted to the ER-to-cytosol retro-translocation process of RTA. In such an artificial system, RTA containing an ER import signal is expressed from an inducible promoter resulting in a suicidal phenotype1,7. Although the cell binding B-subunit of ricin is likewise missing in the experimental setup described in this manuscript and, thus, does not fully represent the natural situation of ricin holotoxin intoxication8, toxin transport from the plasma membrane through the Golgi apparatus to the ER can be closely mimicked with this novel assay. Interestingly, the preliminary results obtained in the pilot study indicate that the trafficking pathways used by RTA reveal striking similarities with the intoxication route of ricin holotoxin.
In summary, the described method can be used to determine the specific role of selected cellular proteins in RTA endocytosis and trafficking in yeast. Furthermore, this experimental setup might be easily adapted to other ribosome inactivating toxins produced and secreted from various yeast and bacterial species such as zymocin or Shiga toxin.

<table>
	<tbody>
		<tr>
			<td>Bacterial and yeast strains</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>E coli BL21 DE3(Gold)</td>
			<td>Aligent Technologies</td>
			<td>230130</td>
			<td></td>
		</tr>
		<tr>
			<td>S. cerevisiae BY4742</td>
			<td>Euroscarf</td>
			<td>Y10000</td>
			<td></td>
		</tr>
		<tr>
			<td>S. cerevisiae BY4742 deletion mutants</td>
			<td>Dharmacon</td>
			<td>YSC1054</td>
			<td>whole collection</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Plasmids used in this protocol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pET24a(+) (Novagen)</td>
			<td>Millipore</td>
			<td>69772-3</td>
			<td></td>
		</tr>
		<tr>
			<td>pET-RTA(His6)</td>
			<td>Becker et al. (2016)3</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Zymolyase 20T</td>
			<td>USBio</td>
			<td>Z1000.250</td>
			<td>lytic enzyme for cell wall removal</td>
		</tr>
		<tr>
			<td>LB broth medium</td>
			<td>Thermo Scientific</td>
			<td>10855021</td>
			<td>15 g agar was added for plate production</td>
		</tr>
		<tr>
			<td>YNB</td>
			<td>Thermo Scientific</td>
			<td>DF0335-15-9</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>A4418-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast drop-out mix supplemts without leucine</td>
			<td>Sigma-Aldrich</td>
			<td>Y1376-20G</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Sigma-Aldrich</td>
			<td>05040-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>D-glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G8270-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Sigma-Aldrich</td>
			<td>10197777001</td>
			<td></td>
		</tr>
		<tr>
			<td>D-raffinose</td>
			<td>Sigma-Aldrich</td>
			<td>83400-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>D-sorbitol</td>
			<td>Sigma-Aldrich</td>
			<td>S1876-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>D-galactose</td>
			<td>Sigma-Aldrich</td>
			<td>G0750-10MG</td>
			<td></td>
		</tr>
		<tr>
			<td>G418</td>
			<td>Thermo Scientific</td>
			<td>11811031</td>
			<td></td>
		</tr>
		<tr>
			<td>IPTG</td>
			<td>Sigma-Aldrich</td>
			<td>I6758-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Roth</td>
			<td>3899.1</td>
			<td></td>
		</tr>
		<tr>
			<td>PAGE ruler prestained</td>
			<td>Fermentas</td>
			<td>26616</td>
			<td>protein ladder used for Western analysis</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Material for RTA purification, desalting and reader measurements</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer Ultrospec 2100 pro</td>
			<td>Amersham</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Soniprep 150</td>
			<td>MSE</td>
			<td></td>
			<td>old model, other models available</td>
		</tr>
		<tr>
			<td>Fluoroskan Ascent</td>
			<td>Thermo Scientific</td>
			<td>5210470</td>
			<td>old model, not available anymore</td>
		</tr>
		<tr>
			<td>&#196;KTAPurifier</td>
			<td>Thermo Scientific</td>
			<td>28406266</td>
			<td>Product is discontinued and replaced</td>
		</tr>
		<tr>
			<td>HisTRAP HP column</td>
			<td>GE Healthcare</td>
			<td>17-5248-02</td>
			<td></td>
		</tr>
		<tr>
			<td>HiTRAP desalting column</td>
			<td>GE Healthcare</td>
			<td>11-0003-29</td>
			<td></td>
		</tr>
		<tr>
			<td>Midisart sterile filter</td>
			<td>Sartorius</td>
			<td>16534K</td>
			<td>0.2 &#181;m pore size</td>
		</tr>
		<tr>
			<td>BCA protein assay kit</td>
			<td>Pierce</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>660 nm assay kit</td>
			<td>Thermo Scientific</td>
			<td>22660</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well plates</td>
			<td>Thermo Scientific</td>
			<td>260860</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Antibodies (optional)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Tetra-His</td>
			<td>Qiagen</td>
			<td>34670</td>
			<td>primary antibody; 1:1,000 dilution</td>
		</tr>
		<tr>
			<td>Anti-mouse-HRP</td>
			<td>Sigma-Aldrich</td>
			<td>A9044-2ML</td>
			<td>secondary antibody, 1:10,000 dilution</td>
		</tr>
	</tbody>
</table>

a simple fluorescence-based reporter assay to identify cellular components required for ricin toxin a chain (rta) trafficking in yeast

Bacterial and plant A/B toxins exploit the natural trafficking pathways in eukaryotic cells to reach their intracellular target(s) in the cytosol and to ultimately kill. Such A/B toxins generally consist of an enzymatically active Asubunit (e.g., ricin toxin A (RTA)) and one or more cell binding Bsubunit(s), which are responsible for toxin binding to specific cell surface receptors. Our current knowledge of how A/B toxins are capable of efficiently intoxicating cells helped scientists to understand fundamental cellular mechanisms, like endocytosis and intracellular protein sorting in higher eukaryotic cells. From a medical point of view, it is likewise important to identify the major toxin trafficking routes to find adequate treatment solutions for patients or to eventually develop therapeutic toxin-based applications for cancer therapy.
Since genome-wide analyses of A/B toxin trafficking in mammalian cells is complex, time-consuming, and expensive, several studies on A/B toxin transport have been performed in the yeast model organism Saccharomyces cerevisiae. Despite being less complex, fundamental cellular processes in yeast and higher eukaryotic cells are similar and very often results obtained in yeast can be transferred to the mammalian situation.
Here, we describe a fast and easy to use reporter assay to analyze the intracellular trafficking of RTA in yeast. An essential advantage of the new assay is the opportunity to investigate not only RTA retro-translocation from the endoplasmic reticulum (ER) into the cytosol, but rather endocytosis and retrograde toxin transport from the plasma membrane into the ER. The assay makes use of a reporter plasmid that allows indirect measurement of RTA toxicity through fluorescence emission of the green fluorescent protein (GFP) after in vivo translation. Since RTA efficiently prevents the initiation of protein biosynthesis by 28S rRNA depurination, this assay allows the identification of host cell proteins involved in intracellular RTA transport through the detection of changes in fluorescence emission.

The general workflow of the protocol described in this manuscript is illustrated in Figure 1, roughly summarizing the single steps for successful RTA purification and the subsequent GFP reporter assay experiment. A more detailed description of each individual step can be found in the protocol. Figure 2 illustrates the expected result of a successful RTA purification by affinity chromatography (Figure 2A</stro...

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A Simple Fluorescence-based Reporter Assay to Identify Cellular Components Required for Ricin Toxin A Chain RTA Trafficking in Yeast

In the manuscript, we describe the use of a yeast-based fluorescence reporter assay to identify cellular components involved in the trafficking and killing processes of the cytotoxic A subunit of the plant toxin ricin (RTA).

A Simple Fluorescence-based Reporter Assay to Identify Cellular Components Required for Ricin Toxin A Chain (RTA) Trafficking in Yeast

Bacterial and plant A/B toxins exploit the natural trafficking pathways in eukaryotic cells to reach their intracellular target(s) in the cytosol and to ...

The overall goal of this simple fluorescence-based reporter assay is to screen for cellular components required for proper trafficking of ricin toxin A or RTA chain in the yeast model organism. This method can help to answer key questions concerning the intercellular transport of ribosome in activating a B toxin. In our case, the cytotoxic subunit of the plain toxin ricin from the plasma membrane into the cytosome.The main advantage of this assay is the opportunity to analyze the effect of an individual yeast gene on RTA transport in a fast and inexpensive way. Begin this protocol with cell transformation as detailed in the text protocol. Inoculate five milliliters of LB kanamycin medium with cells containing RTA expression plasmid or the empty vector.Incubate the cells at 37 degrees Celsius and 220 RPM for 24 hours. Supplement one liter of LB kanamycin medium with five milliliters of preculture. Then incubate the cells at 37 degrees Celsius and 220 RPM until cells have reached an optical density at 600 nanometers of 0.8 to 1.0.Thereafter, reduce the culture temperature to 28 degrees Celsius. Induce RTA expression of the E.Coli by adding IPTG to a final concentration of one millimolar. After 3.5 hours at 28 degrees Celsius and 220 RPM, harvest the cells by centrifugation at 10, 000 g and four degrees Celsius for 10 minutes.Then wash the pellet with five milliliters of binding buffer and pellet at 10, 000 g and four degrees Celsius for 10 minutes. After repeating the wash once, re-suspend the pellet in five milliliters of binding buffer. Sonicate the cells on ice using five cycles of a 15 second pulse and a 30 second pause.After centrifuging the cell licate at 21, 000 g and four degrees Celsius for 15 minutes, filter the supernatant using a sterile syringe filter system. Use an automated purification system, equipped with a five milliliter, nickel-based affinity column to purify the HIS-tagged RTA fraction from the sterile filtered E.Coli supernatant. In general, use an allusion speed of one milliliter per minute and cool the whole purification system to prevent non-efficient toxin binding and loss of toxin activity.Briefly equilibrate the affinity column with 20 milliliters of binding buffer to remove the storage buffer. Then apply the sterile, filtered supernatant onto the affinity column using a syringe. Wash the column with 25 to 35 milliliters of binding buffer to remove the unbound proteins from the column.Perform the washing step until the UV absorbents at 280 nanometers is close to the initial UV value. Then elute the bound RTA fraction with 20 to 35 milliliters of elution buffer and keep the sample on ice. It's important to start the RTA fraction sampling shortly after the UV, 280 value increases and stop the RTA fraction sampling when it reaches the original base line again.To desalt the eluded RTA fraction, replace the affinity column of the purification system with the desalting column and equilibrate the column with 20 milliliters of 0.8 molar sorbitol. Apply the eluded RTA fraction to the column via a syringe. Wash the column with 100 milliliters of 0.8 molar sorbitol and dilute the desalted RTA fraction in a 15 milliliter tube as soon as the UV absorption starts to increase.To avoid salt contamination, stop at the fraction sampling directly when conductance increases. Store the Eluded RTA fraction at four degrees Celsius. After transforming the yeast as described in the text protocol, pick three different yeast clones from each plate.Inoculate the clones in 100 milliliters of lucine dropout raffinose medium at 220 RPM and 30 degrees Celsius to an optical density at 600 nanometers of 1.0 to 2.0. Centrifuge the cells at 25 degrees Celsius for five minutes at 10, 000 g. After washing the cells twice with five milliliters of sterile water, re-suspend the cells in 50 milliliters of spheroplasting buffer.Incubate the 50 milliliter culture at 100 RPM and 30 degrees Celsius for 90 minutes. Following centrifugation at 400 g and four degrees Celsius for 10 minutes, wash the cells twice with five milliliters of stabilized lucine drop out raffinose medium. Then re-suspend the cells in five milliliters of the same medium to use the cells directly for the GFP reporter assay measurements.Seed out the yeast cell spheroplasts into 96 well plates. To each well, add 70 microliters of stabilized lucine drop out raffinose medium, containing the negative control, expressing the empty vector or purified RTA in a final RTA concentration of five micromolar. Add 70 microliters of stabilized lucine drop out medium to each of the remaining wells to bring their total volume to 270 microliters.For a positive control for protein inhibition, add 70 microliters of 0.8 molar sorbitol stabilized G418 solution, as G418 prevents GFP expression in yeast. Immediately add 30 microliters of stabilized galactose solution to induce GFP expression. Prepare an additional negative control by adding 30 microliters of stabilized lucine drop out raffinose medium instead of 30%galactose.After finishing sample preparation, put the 96 well plate in a fluorescence reader and prepare for measurement using the 475, 509 nanometer filter set required for GFP fluorescence detection. Perform measurements at 30 degrees Celsius 120 RPM and a shaking diameter of one millimeter over a time window of 20 hours with measuring intervals of 10 minutes. Shown here is a representative purification graph of the affinity chromatography of RTA and the empty vector control.The blue peak in the black box represents the bound RTA fraction. As shown in the black box, no proteins are eluded from the column in the negative control. Shown here is a desalting diagram of an affinity purified RTA fraction.The diagram shows the separation of the RTA protein fraction and the salt fraction. Coomassie staining results after successful purification and desalting show cell lysate or purified samples of different RTA variants. Unmodified RTA was used in this study.To validate the fluorescence spaced reporter assay, relative GFP fluorescence of wild type yeast spheroplasts was measured under induced and noninduced conditions after 20 hours of GFP induction. Relative GFP fluorescence was also analyzed in the presence of RTA, G418, or the negative control. Relative GFP fluorescence was also measured for selected mutants in which corresponding proteins are known to be involved and not involved in RTA trafficking in yeast.The dotted line represented a significance threshold. Once the RTA expression and purification is mastered, the results of the fluorescence report assay can be received in one day. Additionally, this method paves the way for researchers in the field of ribosome inactivating of B toxins to study their intoxication roots in a eucaryotic model organism yeast.

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JoVE Journal

Biology

7.9K visualizações.  Saarland University. The overall goal of this simple fluorescence-based reporter assay is to screen for cellular components required for proper trafficking of ricin toxin A or RTA chain in the yeast model organism. This method can help to answer key questions concerning the intercellular transport of ribosome in activating a B toxin. In our case, the cytotoxic subunit of the plain toxin ricin from the plasma membrane into the cytosome.The main advantage of this assay is the opportunity to analyze the effec...