Study of shiga toxin B-fragment transport from early endosomes to the golgi apparatus

Shiga toxin (Stx) binds to the cell, and it is transported via endosomes and the Golgi apparatus to the endoplasmic reticulum and cytosol, where it exerts its toxic effect. We have recently shown that Stx activates the tyrosine kinase Syk, which in turn induces clathrin phosphorylation and up-regulates Stx uptake. Here, we show that toxin-induced signaling can also regulate another step in intracellular Stx transport. We demonstrate that transport of Stx to the Golgi apparatus is dependent on the mitogen-activated protein kinase p38. Treatment of cells with chemical inhibitors or small interfering RNA targeting p38 inhibited Stx transport to the Golgi and reduced Stx toxicity. This p38 dependence is specific to Stx, because transport of the related toxin ricin was not affected by p38 inhibition. Stx rapidly activated p38, and recruited it to early endosomes in a Ca2+-dependent manner. Furthermore, agonist-induced oscillations in cytosolic Ca2+levels were inhibited upon Stx stimulation, possibly reflecting Stx-dependent local alterations in cytosolic Ca2+levels. Intracellular transport of Stx is Ca2+dependent, and we provide evidence that Stx activates a signaling cascade involving cross talk between Ca2+and p38, to regulate its trafficking to the Golgi apparatus.

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Study of Shiga toxin b-fragment transport from early/recycling endosomes to the Golgi apparatus

Biology of the Cell ◽

10.1016/s0248-4900(98)80076-2 ◽

1998 ◽

Vol 90 (3) ◽

pp. 288-288

Author(s):

Frédéric Mallard ◽

Danièle Tenza ◽

Claude Antony ◽

Jean Salamero ◽

Bruno Goud ◽

...

Keyword(s):

Golgi Apparatus ◽

Shiga Toxin ◽

Toxin B ◽

Recycling Endosomes

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Targeting of Shiga Toxin B-Subunit to Retrograde Transport Route in Association with Detergent-resistant Membranes

Molecular Biology of the Cell ◽

10.1091/mbc.12.8.2453 ◽

2001 ◽

Vol 12 (8) ◽

pp. 2453-2468 ◽

Cited By ~ 195

Author(s):

Thomas Falguières ◽

Frédéric Mallard ◽

Carole Baron ◽

Daniel Hanau ◽

Clifford Lingwood ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Hela Cells ◽

Shiga Toxin ◽

Secretory Pathway ◽

Retrograde Transport ◽

Toxin B ◽

B Subunit ◽

Early Endosomes ◽

Golgi Network ◽

Triton X

In HeLa cells, Shiga toxin B-subunit is transported from the plasma membrane to the endoplasmic reticulum, via early endosomes and the Golgi apparatus, circumventing the late endocytic pathway. We describe here that in cells derived from human monocytes, i.e., macrophages and dendritic cells, the B-subunit was internalized in a receptor-dependent manner, but retrograde transport to the biosynthetic/secretory pathway did not occur and part of the internalized protein was degraded in lysosomes. These differences correlated with the observation that the B-subunit associated with Triton X-100-resistant membranes in HeLa cells, but not in monocyte-derived cells, suggesting that retrograde targeting to the biosynthetic/secretory pathway required association with specialized microdomains of biological membranes. In agreement with this hypothesis we found that in HeLa cells, the B-subunit resisted extraction by Triton X-100 until its arrival in the target compartments of the retrograde pathway, i.e., the Golgi apparatus and the endoplasmic reticulum. Furthermore, destabilization of Triton X-100-resistant membranes by cholesterol extraction potently inhibited B-subunit transport from early endosomes to thetrans-Golgi network, whereas under the same conditions, recycling of transferrin was not affected. Our data thus provide first evidence for a role of lipid asymmetry in membrane sorting at the interface between early endosomes and the trans-Golgi network.

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Direct Pathway from Early/Recycling Endosomes to the Golgi Apparatus Revealed through the Study of Shiga Toxin B-fragment Transport

The Journal of Cell Biology ◽

10.1083/jcb.143.4.973 ◽

1998 ◽

Vol 143 (4) ◽

pp. 973-990 ◽

Cited By ~ 304

Author(s):

Frédéric Mallard ◽

Claude Antony ◽

Danièle Tenza ◽

Jean Salamero ◽

Bruno Goud ◽

...

Keyword(s):

Golgi Apparatus ◽

Shiga Toxin ◽

Brefeldin A ◽

Adaptor Protein ◽

Endocytic Pathway ◽

Toxin B ◽

Recycling Endosomes ◽

Ultrastructural Studies ◽

Late Endosomes ◽

Rapid Kinetics

Shiga toxin and other toxins of this family can escape the endocytic pathway and reach the Golgi apparatus. To synchronize endosome to Golgi transport, Shiga toxin B-fragment was internalized into HeLa cells at low temperatures. Under these conditions, the protein partitioned away from markers destined for the late endocytic pathway and colocalized extensively with cointernalized transferrin. Upon subsequent incubation at 37°C, ultrastructural studies on cryosections failed to detect B-fragment–specific label in multivesicular or multilamellar late endosomes, suggesting that the protein bypassed the late endocytic pathway on its way to the Golgi apparatus. This hypothesis was further supported by the rapid kinetics of B-fragment transport, as determined by quantitative confocal microscopy on living cells and by B-fragment sulfation analysis, and by the observation that actin- depolymerizing and pH-neutralizing drugs that modulate vesicular transport in the late endocytic pathway had no effect on B-fragment accumulation in the Golgi apparatus. B-fragment sorting at the level of early/recycling endosomes seemed to involve vesicular coats, since brefeldin A treatment led to B-fragment accumulation in transferrin receptor–containing membrane tubules, and since B-fragment colocalized with adaptor protein type 1 clathrin coat components on early/recycling endosomes. Thus, we hypothesize that Shiga toxin B-fragment is transported directly from early/recycling endosomes to the Golgi apparatus. This pathway may also be used by cellular proteins, as deduced from our finding that TGN38 colocalized with the B-fragment on its transport from the plasma membrane to the TGN.

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Efficient transport of Shiga toxin to the Golgi apparatus is dependent on newly synthesized glycosphingolipids

Chemistry and Physics of Lipids ◽

10.1016/j.chemphyslip.2007.06.200 ◽

2007 ◽

Vol 149 ◽

pp. S87

Author(s):

Hilde Raa ◽

Stine Grimmer ◽

Kirsten Sandvig

Keyword(s):

Golgi Apparatus ◽

Shiga Toxin ◽

Efficient Transport

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Switching Shiga Toxin (Stx) Type from Stx2d to Stx2a but Not Stx2c Alters Virulence of Stx-Producing Escherichia coli (STEC) Strain B2F1 in Streptomycin (Str)-Treated Mice

Toxins ◽

10.3390/toxins13010064 ◽

2021 ◽

Vol 13 (1) ◽

pp. 64

Author(s):

Beth A. McNichol ◽

Rebecca A. Bova ◽

Kieron Torres ◽

Lan N. Preston ◽

Angela R. Melton-Celsa

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Oral Administration ◽

Amino Acid Composition ◽

Shiga Toxin ◽

Vero Cells ◽

Toxin B ◽

B Subunit ◽

Intestinal Mucus ◽

Binding Subunit

Shiga toxin (Stx)-producing Escherichia coli (STEC) strain B2F1 produces Stx type 2d, a toxin that becomes more toxic towards Vero cells in the presence of intestinal mucus. STEC that make Stx2d are more pathogenic to streptomycin (Str)-treated mice than most STEC that produce Stx2a or Stx2c. However, purified Stx2d is only 2- or 7-fold more toxic by the intraperitoneal route than Stx2a or Stx2c, respectively. We hypothesized, therefore, that the toxicity differences among Stx2a, Stx2c, and Stx2d occur at the level of delivery from the intestine. To evaluate that hypothesis, we altered the toxin type produced by stx2d+ mouse virulent O91:H21 clinical isolate B2F1 to Stx2a or Stx2c. Because B2F1 encodes two copies of stx2d, we did these studies in a derivative of B2F1 in which stx2d1 was deleted. Although the strains were equivalently virulent to the Str-treated mice at the 1010 dose, the B2F1 strain that produced Stx2a was attenuated relative to the ones that produced Stx2d or Stx2c when administered at 103 CFU/mouse. We next compared the oral toxicities of purified Stx2a, Stx2c, and Stx2d. We found that purified Stx2d is more toxic than Stx2a or Stx2c upon oral administration at 4 µg/mouse. Taken together, these studies suggest that Stx2 toxins are most potent when delivered directly from the bacterium. Furthermore, because Stx2d and Stx2c have the identical amino acid composition in the toxin B subunit, our results indicate that the virulence difference between Stx2a and Stx2d and Stx2c resides in the B or binding subunit of the toxins.

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A System for Production and Rapid Purification of Large Amounts of the Shiga Toxin/Shiga-Like Toxin B Subunit

Molecular Pathogenesis of Gastrointestinal Infections ◽

10.1007/978-1-4684-5982-1_39 ◽

1991 ◽

pp. 299-301

Author(s):

Arthur Donohue-Rolfe ◽

David W. K. Acheson ◽

Gerald T. Keusch ◽

Marcia B. Goldberg ◽

Stephanie A. Boyko ◽

...

Keyword(s):

Shiga Toxin ◽

Toxin B ◽

B Subunit ◽

Rapid Purification

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Shiga toxin–binding site for host cell receptor GPP130 reveals unexpected divergence in toxin-trafficking mechanisms

Molecular Biology of the Cell ◽

10.1091/mbc.e13-01-0057 ◽

2013 ◽

Vol 24 (15) ◽

pp. 2311-2318 ◽

Cited By ~ 28

Author(s):

Somshuvra Mukhopadhyay ◽

Brendan Redler ◽

Adam D. Linstedt

Keyword(s):

Shiga Toxin ◽

Transmembrane Domain ◽

Cell Receptor ◽

Toxin B ◽

Toxin Binding ◽

B Subunit ◽

Host Cell Receptor ◽

Developed World ◽

Trafficking Receptor ◽

Shed Light

Shiga toxicosis is caused by retrograde trafficking of one of three types of Shiga toxin (STx), STx, STx1, or STx2. Trafficking depends on the toxin B subunits, which for STx and STx1 are identical and bind GPP130, a manganese (Mn)-sensitive intracellular trafficking receptor. Elevated Mn down-regulates GPP130, rendering STx/STx1 harmless. Its effectiveness against STx2, however, which is a serious concern in the developed world, is not known. Here we show that Mn-induced GPP130 down-regulation fails to block STx2 trafficking. To shed light on this result, we tested the purified B subunit of STx2 for binding to GPP130 and found that it failed to interact. We then mapped residues at the interface of the GPP130-STx/STx1 complex. In GPP130, binding mapped to a seven-residue stretch in its lumenal stem domain next to the transmembrane domain. This stretch was required for STx/STx1 transport. In STx/STx1, binding mapped to a histidine–asparagine pair on a surface-exposed loop of the toxin B subunit. Significantly, these residues are not conserved in STx2, explaining the lack of effectiveness of Mn against STx2. Together our results imply that STx2 uses an evolutionarily distinct trafficking mechanism and that Mn as a potential therapy should be focused on STx/STx1 outbreaks, which account for the vast majority of cases worldwide.

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