Role of Phospholipase A2 in Retrograde Transport of Ricin

Ricin is a protein toxin classified as a bioterror agent, for which there are no known treatment options available after intoxication. It is composed of an enzymatically active A-chain connected by a disulfide bond to a cell binding B-chain. After internalization by endocytosis, ricin is transported retrogradely to the Golgi and ER, from where the ricin A-chain is translocated to the cytosol where it inhibits protein synthesis and thus induces cell death. We have identified cytoplasmic phospholipase A2 (PLA2) as an important factor in ricin retrograde transport. Inhibition of PLA2 protects against ricin challenge, however the toxin can still be endocytosed and transported to the Golgi. Interestingly, ricin transport from the Golgi to the ER is strongly impaired in response to PLA2 inhibition. Confocal microscopy analysis shows that ricin is still colocalized with the trans-Golgi marker TGN46 in the presence of PLA2 inhibitor, but less is colocalized with the cis-Golgi marker GM130. We propose that PLA2 inhibition results in impaired ricin transport through the Golgi stack, thus preventing it from reaching the ER. Consequently, ricin cannot be translocated to the cytosol to exert its toxic action

Protection against Shiga Toxins

Shiga toxins consist of an A-moiety and five B-moieties able to bind the neutral glycosphingolipid globotriaosylceramide (Gb3) on the cell surface. To intoxicate cells efficiently, the toxin A-moiety has to be cleaved by furin and transported retrogradely to the Golgi apparatus and to the endoplasmic reticulum. The enzymatically active part of the A-moiety is then translocated to the cytosol, where it inhibits protein synthesis and in some cell types induces apoptosis. Protection of cells can be provided either by inhibiting binding of the toxin to cells or by interfering with any of the subsequent steps required for its toxic effect. In this article we provide a brief overview of the interaction of Shiga toxins with cells, describe some compounds and conditions found to protect cells against Shiga toxins, and discuss whether they might also provide protection in animals and humans

Protection against Shiga Toxins

Shiga toxins consist of an A-moiety and five B-moieties able to bind the neutral glycosphingolipid globotriaosylceramide (Gb3) on the cell surface. To intoxicate cells efficiently, the toxin A-moiety has to be cleaved by furin and transported retrogradely to the Golgi apparatus and to the endoplasmic reticulum. The enzymatically active part of the A-moiety is then translocated to the cytosol, where it inhibits protein synthesis and in some cell types induces apoptosis. Protection of cells can be provided either by inhibiting binding of the toxin to cells or by interfering with any of the subsequent steps required for its toxic effect. In this article we provide a brief overview of the interaction of Shiga toxins with cells, describe some compounds and conditions found to protect cells against Shiga toxins, and discuss whether they might also provide protection in animals and humans

Mass spectrometry-based measurements of cyclic adenosine monophosphate in cells, simplified using reversed phase liquid chromatography with a polar characterized stationary phase

3′, 5′ – Cyclic adenosine monophosphate (cAMP) is a ubiquitous second messenger that is involved in many cellular functions and biological processes. In several cell types, cholera toxin will increase the level of cAMP, which mediates toxic effects on cells. In this context, we have developed a fast and simple method based on extraction with 5% trichloroacetic acid (TCA) and quantitation with liquid chromatography-mass tandem spectrometry (LC-MS/MS) for measuring cAMP in cells. A main feature of the LC-MS method was employing a reversed phase C18 column (2.1 mm × 50 mm, 1.6 µm particles) compatible with a 100% aqueous mobile phase, providing retention of the highly polar analyte. Isocratic separations allowed for fast subsequent injections. Negative mode electrospray ionization detection was performed with a triple quadrupole (QqQ)MS. cAMP was extracted from cell samples (~106 cells per well) and spiked with a labelled internal standard, using 200 µL of 5% TCA. The extraction solvent was fully compatible for direct injection onto the reversed phase column. After 10 min incubation, the supernatant was removed, and 10 µL of the supernatant was directly analysed by LC-MS. The method was characterized by the simplicity of the extraction, and the speed (3 min retention time of cAMP), sensitivity (250 pg/mL detection limit), and selectivity (separation from interferences e.g. isomeric compounds) of the LC-MS method, and could be used for quantitation of cAMP in the range 1–500 ng/mL cell extract

Geldanamycin Enhances Retrograde Transport of Shiga Toxin in HEp-2 Cells

<div><p>The heat shock protein 90 (Hsp90) inhibitor geldanamycin (GA) has been shown to alter endosomal sorting, diverting cargo destined for the recycling pathway into the lysosomal pathway. Here we investigated whether GA also affects the sorting of cargo into the retrograde pathway from endosomes to the Golgi apparatus. As a model cargo we used the bacterial toxin Shiga toxin, which exploits the retrograde pathway as an entry route to the cytosol. Indeed, GA treatment of HEp-2 cells strongly increased the Shiga toxin transport to the Golgi apparatus. The enhanced Golgi transport was not due to increased endocytic uptake of the toxin or perturbed recycling, suggesting that GA selectively enhances endosomal sorting into the retrograde pathway. Moreover, GA activated p38 and both inhibitors of p38 or its substrate MK2 partially counteracted the GA-induced increase in Shiga toxin transport. Thus, our data suggest that GA-induced p38 and MK2 activation participate in the increased Shiga toxin transport to the Golgi apparatus.</p></div

Retrograde transport of Shiga toxin and ricin is increased upon Hsp90 inhibition.

<p>HEp-2 cells were treated with 10 μM GA or 1 μM radicicol (Rad) for 30 min before 2 μg/ml Shiga B-sulf2 (A) or 4 μg/ml ricin sulf-1 (B) was added and the incubation continued for 1.5 h. The amount of sulfated toxin and the total protein sulfation was determined as described in Materials and Methods. The toxin sulfation (black bars) and total protein sulfation (grey bars) are expressed relative to control treatment (DMSO) and are plotted as mean values + SEM, <i>n</i> ≥ 3. * p ≤ 0.05, *** p ≤ 0.005, paired Student’s <i>t</i>-test.</p

GA enhances CI-M6PR localization to the Golgi.

<p>(A) HeLa cells stably expressing CD8-M6PR fusion protein were treated with 10 μM GA for 30 min at 37°C before they were chilled and incubated with 10 μg/ml CD8 antibody at 4°C for 30 min. The CD8 antibody chase was performed for 0 or 15 min at 37°C. Subsequently, cells were fixed, permeabilized and stained with antibodies against CD8 (magenta) and giantin (green). DAPI is shown in blue. Scale bar 20 μm. (B) The amount of CI-M6PR that has reached the Golgi was quantified as the CI-M6PR intensity in giantin-positive structures relative to the intensity of total cell-associated CI-M6PR in individual cells using Fiji software. The data was normalized to control samples (DMSO) in individual experiments and plotted as mean values + SEM. <i>n</i> = 3, with at least 30–50 cells quantified for each condition.</p

MK2 activation contributes to the increased retrograde transport after GA treatment.

<p>(A) HEp-2 cells were starved in sulfate-free medium for 3 h at 37°C, and during the last 30 min 2.5 μM Akt inhibitor VIII (Akt-i), 2.5 μM PF 3644022 (PF) or 10 μM GA were added. The cells were lysed and proteins were separated by SDS-PAGE. Blots were probed with the indicated antibodies, and Hsp90 was used as a loading control. (B) Cells were preincubated with 10 μM GA in combination with 2.5 μM Akt inhibitor VIII (Akt-i), or 2.5 μM PF 3644022 (PF) for 30 min and subsequently incubated with Shiga B-sulf2 for 1 h. The Shiga B sulfation (black bars) and total protein sulfation (grey bars) are expressed relative to control treatment (DMSO) and are plotted as mean values + SEM, <i>n</i> = 6. * p ≤ 0.05, paired Student’s <i>t</i>-test.</p

GA enhances Shiga toxin localization to the Golgi.

<p>(A) HEp-2 cells were treated with 10 μM GA for 30 min before ~100 ng/ml Shiga toxin 1 mutant was added and the incubation continued for 30 min. Subsequently, cells were fixed, permeabilized and stained with antibodies against Shiga toxin (magenta) and giantin (green). DAPI is shown in blue. Scale bar 20 μm. (B) The amount of Shiga toxin 1 mutant that has reached the Golgi was quantified as the Shiga toxin 1 mutant intensity in giantin-positive structures relative to the intensity of total cell-associated Shiga toxin 1 mutant in individual cells using Fiji software and plotted as mean values + SEM. <i>n</i> = 4, with at least 65 cells quantified for each condition. * p ≤0.05, paired Student’s <i>t</i>-test.</p

Shiga toxin endocytosis is not increased by GA treatment.

<p>HEp-2 cells were preincubated with 10 μM GA for 30 min at 37°C and subsequently incubated with 40 ng/ml biotinylated Shiga toxin 1 mutant for 20 min. The amount of internalized or total cell-associated toxin was quantified as described in Materials and Methods. Mean values + SEM of total cell-associated (black bars) and internalized (grey bars) Shiga toxin 1 mutant are presented as percentage of control (DMSO), <i>n</i> = 3. * p ≤ 0.05 paired Student’s <i>t</i>-test.</p