scholarly journals How Ricin and Shiga Toxin Reach the Cytosol of Target Cells: Retrotranslocation from the Endoplasmic Reticulum

2011 ◽  
pp. 19-40 ◽  
Author(s):  
Robert A. Spooner ◽  
J. Michael Lord
Keyword(s):  
Endoplasmic Reticulum ◽  
Shiga Toxin ◽  
Target Cells

scholarly journals Myosin Motors and Not Actin Comets Are Mediators of the Actin-based Golgi-to-Endoplasmic Reticulum Protein Transport

2003 ◽  
Vol 14 (2) ◽  
pp. 445-459 ◽  
Author(s):  
Juan M. Durán ◽  
Ferran Valderrama ◽  
Susana Castel ◽  
Juana Magdalena ◽  
Mónica Tomás ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Complex ◽  
Light Chain ◽  
Shiga Toxin ◽  
Actin Filaments ◽  
Protein Transport ◽  
Myosin Ii ◽  
Nonmuscle Myosin ◽  
Nonmuscle Myosin Ii ◽  
Myosin Motors

We have previously reported that actin filaments are involved in protein transport from the Golgi complex to the endoplasmic reticulum. Herein, we examined whether myosin motors or actin comets mediate this transport. To address this issue we have used, on one hand, a combination of specific inhibitors such as 2,3-butanedione monoxime (BDM) and 1-[5-isoquinoline sulfonyl]-2-methyl piperazine (ML7), which inhibit myosin and the phosphorylation of myosin II by the myosin light chain kinase, respectively; and a mutant of the nonmuscle myosin II regulatory light chain, which cannot be phosphorylated (MRLC2AA). On the other hand, actin comet tails were induced by the overexpression of phosphatidylinositol phosphate 5-kinase. Cells treated with BDM/ML7 or those that express the MRLC2AA mutant revealed a significant reduction in the brefeldin A (BFA)-induced fusion of Golgi enzymes with the endoplasmic reticulum (ER). This delay was not caused by an alteration in the formation of the BFA-induced tubules from the Golgi complex. In addition, the Shiga toxin fragment B transport from the Golgi complex to the ER was also altered. This impairment in the retrograde protein transport was not due to depletion of intracellular calcium stores or to the activation of Rho kinase. Neither the reassembly of the Golgi complex after BFA removal nor VSV-G transport from ER to the Golgi was altered in cells treated with BDM/ML7 or expressing MRLC2AA. Finally, transport carriers containing Shiga toxin did not move into the cytosol at the tips of comet tails of polymerizing actin. Collectively, the results indicate that 1) myosin motors move to transport carriers from the Golgi complex to the ER along actin filaments; 2) nonmuscle myosin II mediates in this process; and 3) actin comets are not involved in retrograde transport.

Comparison of a New Enrichment Procedure for Shiga Toxin–Producing Escherichia coli with Five Standard Methods

2005 ◽  
Vol 68 (8) ◽  
pp. 1593-1599 ◽  
Author(s):  
MICHAEL A. GRANT
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Shiga Toxin ◽  
Real Time Pcr ◽  
Target Cells ◽  
Macconkey Agar ◽  
Standard Methods ◽  
E Coli ◽  
Canadian Health ◽  
Health Products

A new procedure for enrichment of Escherichia coli O157:H7 and other Shiga toxin–producing E. coli was compared to five standard methods: the British Public Health Laboratory Service, International Standard Method, U.S. Department of Agriculture, Canadian Health Products and Food Branch, and U.S. Food and Drug Administration. The new procedure was comparable to the standard methods in its ability to detect target cells inoculated into foods at approximately 1 CFU g−1. Comparisons were also made of the ability of the six enrichment procedures to detect E. coli O157:H7 against a large background of competitor microorganisms. In these experiments the new procedure yielded more target cells than the other five enrichments by two to three orders of magnitude as determined by enumeration on sorbitol MacConkey agar with tellurite and cefixime and Rainbow agar with tellurite and novobiocin and by verification of presumptive colonies by real-time PCR. For example, the population of enterohemorrhagic E. coli strain 6341 recovered on sorbitol MacConkey agar with tellurite and cefixime after enrichment with the experimental method was 2.42 × 108 CFU ml−1 and 1.80 × 106 CFU ml−1 after enrichment with the Canadian Health Products and Food Branch method, the second most effective in this experiment. In addition, broth cultures resulting from each of the six enrichment procedures were used to prepare templates for real-time PCR detection of stx1/stx2. Resulting threshold cycle (Ct) values after the experimental enrichment were similar to positive control values, whereas the five standard methods produced delayed Ct values or were not detected.

Functional Organization of the Endoplasmic Reticulum Dictates the Susceptibility of Target Cells to Arsenite-Induced Mitochondrial Superoxide Formation, Mitochondrial Dysfunction and Apoptosis

2021 ◽  
Author(s):  
Andrea Guidarelli ◽  
Alessia Catalani ◽  
Ersilia Varone ◽  
Stefano Fumagalli ◽  
Ester Zito ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Mitochondrial Dysfunction ◽  
Functional Organization ◽  
Close Contact ◽  
Cell Types ◽  
Target Cells ◽  
Close Apposition ◽  
Mitochondrial Superoxide ◽  
Low Concentrations ◽  
Absolute Requirement

Abstract Arsenite induces many critical effects associated with the formation of reactive oxygen species (ROS) through different mechanisms. We focused on the Ca2+-dependent mitochondrial superoxide (mitoO2-.) formation and addressed questions on the effects of low concentrations of arsenite on the mobilization of the cation from the endoplasmic reticulum and the resulting mitochondrial accumulation. Using various differentiated and undifferentiated cell types uniquely expressing the inositol-1, 4, 5-triphosphate receptor (IP3R), or both the IP3R and the ryanodine receptor (RyR), we determined that expression of this second Ca2+ channel is an absolute requirement for mitoO2-. formation and for the ensuing mitochondrial dysfunction and downstream apoptosis. In arsenite-treated cells, RyR was recruited after IP3R stimulation and agonist studies indicated that in these cells RyR is in close apposition with mitochondria. It was also interesting to observe that arsenite fails to promote mitochondrial Ca2+ accumulation, mitoO2-. formation, mitochondrial toxicity in RyR-devoid cells, in which the IP3R is in close contact with the mitochondria. We therefore conclude that low dose arsenite-induced mitoO2- formation and the resulting mitochondrial dysfunction and toxicity, are prerequisite of cell types expressing the RyR in close apposition with mitochondria.

scholarly journals In Vivo Tumor Targeting by the B-Subunit of Shiga Toxin

2008 ◽  
Vol 7 (6) ◽  
pp. 7290.2008.00022 ◽  
Author(s):  
Thomas Viel ◽  
Estelle Dransart ◽  
Fariba Nemati ◽  
Emilie Henry ◽  
Benoit Thézé ◽  
...  
Keyword(s):  
Shiga Toxin ◽  
Renal Excretion ◽  
Cellular Level ◽  
Target Cells ◽  
B Subunit ◽  
Monocytes And Macrophages ◽  
Intestinal Pathogen ◽  
Tumoral Cells ◽  
Tumor Area

Delivery of drugs to the appropriate target cells would improve efficacy and reduce potential side effects. The nontoxic B-subunit of the intestinal pathogen-produced Shiga toxin (STxB) binds specifically to the glycosphingolipid Gb3, overex-pressed in membranes of certain tumor cells, and enters these cells through the retrograde pathway. Therefore, STxB binding to Gb3 receptors may be useful for cell-specific vectorization or imaging purposes. Here we labeled STxB with a fluorophore to evaluate its potential as an in vivo cell-specific targeting reagent in two different models of human colorectal carcinoma. Fluorescent STxB was administered systemically to xenografted nude mice, and its biodistribution was studied by optical imaging. The use of fluorescent STxB allowed the combination of the macroscopic observations with analyses at the cellular level using confocal microscopy. After administration, the fluorescent STxB was slowly eliminated by renal excretion. However, it accumulated in the tumor area. Furthermore, STxB was demonstrated to enter the Gb3-expressing tumoral cells, as well as the epithelial cells of the neovascularization and the monocytes and macrophages surrounding the xenografts.

scholarly journals Pathogenic Mechanism of Mouse Brain Damage Caused by Oral Infection with Shiga Toxin-Producing Escherichia coliO157:H7

2000 ◽  
Vol 68 (3) ◽  
pp. 1207-1214 ◽  
Author(s):  
Eiji Kita ◽  
Yoshihisa Yunou ◽  
Takaaki Kurioka ◽  
Hiroko Harada ◽  
Shinji Yoshikawa ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Brain Damage ◽  
Shiga Toxin ◽  
Vascular Endothelial Cells ◽  
Oral Infection ◽  
Target Cells ◽  
Vascular Endothelial ◽  
Short Term Exposure ◽  
Tnf Α ◽  
The Brain

ABSTRACT In a previous study, we showed that infection with Shiga toxin (Stx)-producing Escherichia coli O157:H7 (strain SmrN-9) caused neurologic symptoms in malnourished mice with positive immunoreactions of Stx2 in brain tissues. The present study explores the mechanism of how Stx injures the vascular endothelium to enter the central nervous system in mice. Oral infection with strain SmrN-9 elicited a tumor necrosis factor alpha (TNF-α) response in the blood as early as 2 days after infection, while Stx was first detected at 3 days postinfection. In the brain, TNF-α was detected at day 3, and its quantity was increased over the next 3 days. Frozen sections of the brains from moribound mice contained high numbers of apoptotic cells. Glycolipids recognized by an anti-Gb3 monoclonal antibody were extracted from the brain, and purified Stx2 was able to bind to the glycolipids. In human umbilical vascular endothelial cells (HUVEC) cultured with fluorescein-labeled Stx2 (100 ng/ml), TNF-α (20 U/ml) significantly facilitated the intracellular compartmentalization of fluorescence during 24 h of incubation, suggesting the enhanced intracellular processing of Stx2. Consequently, higher levels of apoptosis in HUVEC were found at 48 h. Short-term exposure of HUVEC to Stx2 abrogated their apoptotic response to subsequent incubation with TNF-α alone or TNF-α and Stx2. In contrast, primary exposure of HUVEC to TNF-α followed by exposure to Stx2 alone or TNF-α and Stx2 induced apoptosis at the same level as obtained after 48-h incubation with these two agents. These results suggest that the rapid production of circulating TNF-α after infection induces a state of competence in vascular endothelial cells to undergo apoptosis, which would be finally achieved by subsequent elevation of Stx in the blood. In this synergistic action, target cells must be first exposed to TNF-α. Such cell injury may be a prerequisite to brain damage after infection with Stx-producing E. coliO157:H7.

scholarly journals Shiga Toxin 2-Induced Endoplasmic Reticulum Stress Is Minimized by Activated Protein C but Does Not Correlate with Lethal Kidney Injury

Toxins ◽  
2015 ◽  
Vol 7 (1) ◽  
pp. 170-186 ◽  
Author(s):  
Caitlin Parello ◽  
Chad Mayer ◽  
Benjamin Lee ◽  
Amanda Motomochi ◽  
Shinichiro Kurosawa ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Endoplasmic Reticulum Stress ◽  
Shiga Toxin ◽  
Protein C ◽  
Activated Protein C ◽  
Kidney Injury ◽  
Shiga Toxin 2

scholarly journals Bovine Ileal Intraepithelial Lymphocytes Represent Target Cells for Shiga Toxin 1 from Escherichia coli

2004 ◽  
Vol 72 (4) ◽  
pp. 1896-1905 ◽  
Author(s):  
Christian Menge ◽  
Maike Blessenohl ◽  
Tobias Eisenberg ◽  
Ivonne Stamm ◽  
Georg Baljer
Keyword(s):  
Escherichia Coli ◽  
T Cells ◽  
Shiga Toxin ◽  
Killer Cell ◽  
Cell Activity ◽  
Intraepithelial Lymphocytes ◽  
Reservoir Host ◽  
Interleukin 4 ◽  
Target Cells ◽  
Adult Cattle

ABSTRACT The discovery that bovine peripheral lymphocytes are sensitive to Stx1 identified a possible mechanism for the persistence of infections with Shiga toxin (Stx)-producing Escherichia coli (STEC) in the bovine reservoir host. If intraepithelial lymphocytes (IEL) are also sensitive to Stx1, the idea that Stx1 affects inflammation in the bovine intestine is highly attractive. To prove this hypothesis, ileal IEL (iIEL) were prepared from adult cattle, characterized by flow cytometry, and subjected to functional assays in the presence and absence of purified Stx1. We found that 14.9% of all iIEL expressed Gb3/CD77, the Stx1 receptor on bovine lymphocytes, and 7.9% were able to bind the recombinant B subunit of Stx1. The majority of Gb3/CD77+ cells were activated CD3+ CD6+ CD8α+ T cells, whereas only some CD4+ T cells and B cells expressed Gb3/CD77. However, Stx1 blocked the mitogen-induced transformation to enlarged blast cells within all subpopulations to a similar extent and significantly reduced the percentage of Gb3/CD77+ cells. Although Stx1 did not affect the natural killer cell activity of iIEL, the toxin accelerated the synthesis of interleukin-4 (IL-4) mRNA and reduced the amount of IL-8 mRNA in bovine iIEL cultures. Because the intestinal system comprises a rich network of interactions between different types of cells and any dysfunction may influence the course of intestinal infections, this demonstration that Stx1 can target bovine IEL may be highly relevant for our understanding of the interplay between STEC and its reservoir host.

scholarly journals AB5Preassembly Is Not Required for Shiga Toxin Activity

2016 ◽  
Vol 198 (11) ◽  
pp. 1621-1630 ◽  
Author(s):  
Christine A. Pellino ◽  
Sayali S. Karve ◽  
Suman Pradhan ◽  
Alison A. Weiss
Keyword(s):  
Escherichia Coli ◽  
Enzymatic Activity ◽  
Hemolytic Uremic Syndrome ◽  
Shiga Toxin ◽  
Target Cells ◽  
Content Type ◽  
B Subunit ◽  
Uremic Syndrome ◽  
A Subunit ◽  
B Subunits

ABSTRACTShiga toxin (Stx)-producingEscherichia coli(STEC) is a major cause of foodborne illness, including the life-threatening complication hemolytic-uremic syndrome. The German outbreak in 2011 resulted in nearly 4,000 cases of infection, with 54 deaths. Two forms of Stx, Stx1 and Stx2, differ in potency, and subtype Stx2a is most commonly associated with fatal human disease. Stx is considered to be an AB5toxin. The single A (enzymatically active) subunit inhibits protein synthesis by cleaving a catalytic adenine from the eukaryotic rRNA. The B (binding) subunit forms a homopentamer and mediates cellular association and toxin internalization by binding to the glycolipid globotriaosylceramide (Gb3). Both subunits are essential for toxicity. Here we report that unlike other AB5toxin family members, Stx is produced by STEC as unassembled A and B subunits. A preformed AB5complex is not required for cellular toxicity orin vivotoxicity to mice, and toxin assembly likely occurs at the cell membrane. We demonstrate that disruption of A- and B-subunit association by use of A-subunit peptides that lack enzymatic activity can protect mice from lethal doses of toxin. Currently, no treatments have been proven to be effective for hemolytic-uremic syndrome. Our studies demonstrate that agents that interfere with A- and B-subunit assembly may have therapeutic potential. Shiga toxin (Stx) produced by pathogenicEscherichia coliis considered to be an AB5heterohexamer; however, no known mechanisms ensure AB5assembly. Stx released byE. coliis not in the AB5conformation and assembles at the receptor interface. Thus, unassembled Stx can impart toxicity. This finding shows that preventing AB5assembly is a potential treatment for Stx-associated illnesses.IMPORTANCEComplications due to Shiga toxin are frequently fatal, and at present, supportive care is the only treatment option. Furthermore, antibiotic treatment is contraindicated due to the ability of antibiotics to amplify bacterial expression of Shiga toxin. We report, contrary to prevailing assumptions, that Shiga toxin produced by STEC circulates as unassembled A and B subunits at concentrations that are lethal to mice. Similar to the case for anthrax toxin, assembly occurs on receptors expressed on the surfaces of mammalian target cells. Disruption of Shiga toxin assembly by use of A-subunit peptides that lack enzymatic activity protects mice from lethal challenge with Shiga toxin, suggesting a new approach for development of therapeutics.

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