scholarly journals Shiga Toxin Facilitates Its Retrograde Transport by Modifying Microtubule Dynamics

2006 ◽  
Vol 17 (10) ◽  
pp. 4379-4389 ◽  
Author(s):  
Heidi Hehnly ◽  
David Sheff ◽  
Mark Stamnes
Keyword(s):  
Shiga Toxin ◽  
Intracellular Transport ◽  
Secretory Pathway ◽  
Retrograde Transport ◽  
Host Cells ◽  
Mammalian Host ◽  
Golgi Stack ◽  
Toxin Binding ◽  
Cytoskeletal Dynamics ◽  
Induced Changes

The bacterial exotoxin Shiga toxin is endocytosed by mammalian host cells and transported retrogradely through the secretory pathway before entering the cytosol. Shiga toxin also increases the levels of microfilaments and microtubules (MTs) upon binding to the cell surface. The purpose for this alteration in cytoskeletal dynamics is unknown. We have investigated whether Shiga toxin-induced changes in MT levels facilitate its intracellular transport. We have tested the effects of the Shiga toxin B subunit (STB) on MT-dependent and -independent transport steps. STB increases the rate of MT-dependent Golgi stack repositioning after nocodazole treatment. It also enhances the MT-dependent accumulation of transferrin in a perinuclear recycling compartment. By contrast, the rate of MT-independent transferrin recycling is not significantly different when STB is present. We found that STB normally requires MTs and dynein for its retrograde transport to the juxtanuclear Golgi complex and that STB increases MT assembly. Furthermore, we find that MT polymerization is limiting for STB transport in cells. These results show that STB-induced changes in cytoskeletal dynamics influence intracellular transport. We conclude that the increased rate of MT assembly upon Shiga toxin binding facilitates the retrograde transport of the toxin through the secretory pathway.

scholarly journals Retrograde Shiga Toxin Trafficking Is Regulated by ARHGAP21 and Cdc42

2009 ◽  
Vol 20 (20) ◽  
pp. 4303-4312 ◽  
Author(s):  
Heidi Hehnly ◽  
Katrina Marie Longhini ◽  
Ji-Long Chen ◽  
Mark Stamnes
Keyword(s):  
Golgi Apparatus ◽  
Shiga Toxin ◽  
Rho Gtpases ◽  
Secretory Pathway ◽  
Retrograde Transport ◽  
Protein Translation ◽  
Dependent Manner ◽  
Intestinal Epithelial ◽  
Food Borne ◽  
Cytoskeletal Dynamics

Shiga-toxin–producing Escherichia coli remain a food-borne health threat. Shiga toxin is endocytosed by intestinal epithelial cells and transported retrogradely through the secretory pathway. It is ultimately translocated to the cytosol where it inhibits protein translation. We found that Shiga toxin transport through the secretory pathway was dependent on the cytoskeleton. Recent studies reveal that Shiga toxin activates signaling pathways that affect microtubule reassembly and dynein-dependent motility. We propose that Shiga toxin alters cytoskeletal dynamics in a way that facilitates its transport through the secretory pathway. We have now found that Rho GTPases regulate the endocytosis and retrograde motility of Shiga toxin. The expression of RhoA mutants inhibited endocytosis of Shiga toxin. Constitutively active Cdc42 or knockdown of the Cdc42-specific GAP, ARHGAP21, inhibited the transport of Shiga toxin to the juxtanuclear Golgi apparatus. The ability of Shiga toxin to stimulate microtubule-based transferrin transport also required Cdc42 and ARHGAP21 function. Shiga toxin addition greatly decreases the levels of active Cdc42-GTP in an ARHGAP21-dependent manner. We conclude that ARHGAP21 and Cdc42-based signaling regulates the dynein-dependent retrograde transport of Shiga toxin to the Golgi apparatus.

scholarly journals Blood group P1 antigen–bearing glycoproteins are functional but less efficient receptors of Shiga toxin than conventional glycolipid-based receptors

2020 ◽  
Vol 295 (28) ◽  
pp. 9490-9501 ◽  
Author(s):  
Kanta Morimoto ◽  
Noriko Suzuki ◽  
Isei Tanida ◽  
Soichiro Kakuta ◽  
Yoko Furuta ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Death ◽  
Blood Group ◽  
Shiga Toxin ◽  
Retrograde Transport ◽  
Host Cells ◽  
Mammalian Host ◽  
B Subunit ◽  
Early Endosomes ◽  
Hydrophobic Portion

Shiga toxin (STx) is a virulence factor produced by enterohemorrhagic Escherichia coli. STx is taken up by mammalian host cells by binding to the glycosphingolipid (GSL) globotriaosylceramide (Gb3; Galα1-4Galβ1-4Glc-ceramide) and causes cell death after its retrograde membrane transport. However, the contribution of the hydrophobic portion of Gb3 (ceramide) to STx transport remains unclear. In pigeons, blood group P1 glycan antigens (Galα1-4Galβ1-4GlcNAc-) are expressed on glycoproteins that are synthesized by α1,4-galactosyltransferase 2 (pA4GalT2). To examine whether these glycoproteins can also function as STx receptors, here we constructed glycan-remodeled HeLa cell variants lacking Gb3 expression but instead expressing pA4GalT2-synthesized P1 glycan antigens on glycoproteins. We compared STx binding and sensitivity of these variants with those of the parental, Gb3-expressing HeLa cells. The glycan-remodeled cells bound STx1 via N-glycans of glycoproteins and were sensitive to STx1 even without Gb3 expression, indicating that P1-containing glycoproteins also function as STx receptors. However, these variants were significantly less sensitive to STx than the parent cells. Fluorescence microscopy and correlative light EM revealed that the STx1 B subunit accumulates to lower levels in the Golgi apparatus after glycoprotein-mediated than after Gb3-mediated uptake but instead accumulates in vacuole-like structures probably derived from early endosomes. Furthermore, coexpression of Galα1-4Gal on both glycoproteins and GSLs reduced the sensitivity of cells to STx1 compared with those expressing Galα1-4Gal only on GSLs, probably because of competition for STx binding or internalization. We conclude that lipid-based receptors are much more effective in STx retrograde transport and mediate greater STx cytotoxicity than protein-based receptors.

scholarly journals ER/Golgi Intermediates Acquire Golgi Enzymes by Brefeldin a–Sensitive Retrograde Transport in Vitro

1999 ◽  
Vol 147 (7) ◽  
pp. 1457-1472 ◽  
Author(s):  
Chung-Chih Lin ◽  
Harold D. Love ◽  
Jennifer N. Gushue ◽  
John J.M. Bergeron ◽  
Joachim Ostermann
Keyword(s):  
Secretory Pathway ◽  
Mutant Cell ◽  
Brefeldin A ◽  
Retrograde Transport ◽  
Secretory Proteins ◽  
Intermediate Step ◽  
Golgi Stack ◽  
Transport Vesicles ◽  
Direct Fusion

Secretory proteins exit the ER in transport vesicles that fuse to form vesicular tubular clusters (VTCs) which move along microtubule tracks to the Golgi apparatus. Using the well-characterized in vitro approach to study the properties of Golgi membranes, we determined whether the Golgi enzyme NAGT I is transported to ER/Golgi intermediates. Secretory cargo was arrested at distinct steps of the secretory pathway of a glycosylation mutant cell line, and in vitro complementation of the glycosylation defect was determined. Complementation yield increased after ER exit of secretory cargo and was optimal when transport was blocked at an ER/Golgi intermediate step. The rapid drop of the complementation yield as secretory cargo progresses into the stack suggests that Golgi enzymes are preferentially targeted to ER/Golgi intermediates and not to membranes of the Golgi stack. Two mechanisms for in vitro complementation could be distinguished due to their different sensitivities to brefeldin A (BFA). Transport occurred either by direct fusion of preexisting transport intermediates with ER/Golgi intermediates, or it occurred as a BFA-sensitive and most likely COP I–mediated step. Direct fusion of ER/Golgi intermediates with cisternal membranes of the Golgi stack was not observed under these conditions.

scholarly journals Retrograde transport from the Golgi region to the endoplasmic reticulum is sensitive to GTP gamma S.

1992 ◽  
Vol 116 (6) ◽  
pp. 1357-1367 ◽  
Author(s):  
A Tan ◽  
J Bolscher ◽  
C Feltkamp ◽  
H Ploegh
Keyword(s):  
Hepg2 Cells ◽  
Intracellular Transport ◽  
Secretory Pathway ◽  
Binding Proteins ◽  
Brefeldin A ◽  
Retrograde Transport ◽  
Gtp Binding Proteins ◽  
Gtp Binding ◽  
Streptolysin O ◽  
Gamma S

The involvement of GTP-binding proteins in the intracellular transport of the secretory glycoprotein alpha 1-antitrypsin was investigated in streptolysin O-permeabilized HepG2 cells. This permeabilization procedure allows ready access to the intracellular milieu of the membrane-impermeant, nonhydrolyzable GTP analog GTP gamma S. In streptolysin O-permeabilized HepG2 cells, the constitutive secretory pathway remains functional and is sensitive to GTP gamma S. Exposure of HepG2 cells to brefeldin A resulted in redistribution of Golgi-resident glycosyltransferases (including both alpha 2----3 and alpha 2----6 sialyltransferases) to the ER. This redistribution was sensitive to GTP gamma S. Our results suggest that GTP-binding proteins are involved in the regulation not only of the anterograde, but also of the retrograde, pathway.

Endocytosis, intracellular transport, and cytotoxic action of Shiga toxin and ricin

1996 ◽  
Vol 76 (4) ◽  
pp. 949-966 ◽  
Author(s):  
K. Sandvig ◽  
B. van Deurs
Keyword(s):  
Shiga Toxin ◽  
Intracellular Transport ◽  
Retrograde Transport ◽  
Cytotoxic Action ◽  
Coated Pits ◽  
Polarized Epithelia ◽  
Protein Toxins ◽  
A Cell ◽  
Membrane Bound ◽  
Golgi Network

Protein toxins such as ricin and Shiga toxin with intracellular targets have to be endocytosed and translocated to the cytosol to inhibit the protein synthesis and thereby kill the cell. Ricin is internalized by both clathrin-dependent and -independent endocytic mechanisms, whereas Shiga toxin seems to be taken up exclusively from clathrin-coated pits. After endocytosis, internalized membrane and content are delivered to endosomes, where sorting for further routing in the cell takes place. Toxins that remain membrane bound at low endosomal pH can be recycled to the cell surface or transcytosed in polarized epithelia. A large proportion of internalized toxin is transported to lysosomes for degradation. Most importantly, a fraction of the internalized ricin and Shiga toxin molecules is delivered to the trans-Golgi network (TGN). Shiga toxin can, in some very sensitive cells, be transported retrogradely through the Golgi cisterns all the way back to the endoplasmic reticulum (ER), and it is possible that also ricin is transported retrogradely to the ER. In this review, a cell biological overview of these intracellular transport steps is presented, and evidence is provided that the delivery to the TGN and the subsequent retrograde transport to the ER are required for optimal intoxication. Moreover, it is argued that knowledge of this transport is important for targeted drug delivery such as the application of immunotoxins in cancer therapy.

I. Shiga toxin B-subunit system: retrograde transport, intracellular vectorization, and more

2002 ◽  
Vol 283 (1) ◽  
pp. G1-G7 ◽  
Author(s):  
Ludger Johannes
Keyword(s):  
Endoplasmic Reticulum ◽  
Shiga Toxin ◽  
Intracellular Transport ◽  
Molecular Mechanisms ◽  
Physiological Function ◽  
Retrograde Transport ◽  
Toxin B ◽  
B Subunit ◽  
New Methods ◽  
Therapeutic Compounds

Many intracellular transport routes are still little explored. This is particularly true for retrograde transport between the plasma membrane and the endoplasmic reticulum. Shiga toxin B subunit has become a powerful tool to study this pathway, and recent advances on the molecular mechanisms of transport in the retrograde route and on its physiological function(s) are summarized. Furthermore, it is discussed how the study of retrograde transport of Shiga toxin B subunit allows one to design new methods for the intracellular delivery of therapeutic compounds.

scholarly journals Rab6 Coordinates a Novel Golgi to ER Retrograde Transport Pathway in Live Cells

1999 ◽  
Vol 147 (4) ◽  
pp. 743-760 ◽  
Author(s):  
Jamie White ◽  
Ludger Johannes ◽  
Frédéric Mallard ◽  
Andreas Girod ◽  
Stephan Grill ◽  
...  
Keyword(s):  
Shiga Toxin ◽  
Secretory Pathway ◽  
Fluorescent Protein ◽  
Retrograde Transport ◽  
Live Cells ◽  
Cell Periphery ◽  
Wild Type ◽  
Transport Pathway ◽  
Toxin B ◽  
Kdel Receptor

We visualized a fluorescent-protein (FP) fusion to Rab6, a Golgi-associated GTPase, in conjunction with fluorescent secretory pathway markers. FP-Rab6 defined highly dynamic transport carriers (TCs) translocating from the Golgi to the cell periphery. FP-Rab6 TCs specifically accumulated a retrograde cargo, the wild-type Shiga toxin B-fragment (STB), during STB transport from the Golgi to the endoplasmic reticulum (ER). FP-Rab6 TCs associated intimately with the ER, and STB entered the ER via specialized peripheral regions that accumulated FP-Rab6. Microinjection of antibodies that block coatomer protein I (COPI) function inhibited trafficking of a KDEL-receptor FP-fusion, but not FP-Rab6. Additionally, markers of COPI-dependent recycling were excluded from FP-Rab6/STB TCs. Overexpression of Rab6:GDP (T27N mutant) using T7 vaccinia inhibited toxicity of Shiga holotoxin, but did not alter STB transport to the Golgi or Golgi morphology. Taken together, our results indicate Rab6 regulates a novel Golgi to ER transport pathway.

scholarly journals Bacteria-Host Crosstalk: Sensing of the Quorum in the Context of Pseudomonas aeruginosa Infections

2018 ◽  
Vol 11 (3) ◽  
pp. 263-279 ◽  
Author(s):  
Maria V. Turkina ◽  
Elena Vikström
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Current Knowledge ◽  
Single Species ◽  
Opportunistic Pathogen ◽  
Host Cells ◽  
Future Perspective ◽  
Mammalian Host ◽  
Acylhomoserine Lactone ◽  
Cytoskeletal Dynamics ◽  
Biofilm Development

Cell-to-cell signaling via small molecules is an essential process to coordinate behavior in single species within a community, and also across kingdoms. In this review, we discuss the quorum sensing (QS) systems used by the opportunistic pathogen Pseudomonas aeruginosa to sense bacterial population density and fitness, and regulate virulence, biofilm development, metabolite acquisition, and mammalian host defense. We also focus on the role of N-acylhomoserine lactone-dependent QS signaling in the modulation of innate immune responses connected together via calcium signaling, homeostasis, mitochondrial and cytoskeletal dynamics, and governing transcriptional and proteomic responses of host cells. A future perspective emphasizes the need for multidisciplinary efforts to bring current knowledge of QS into a more detailed understanding of the communication between bacteria and host, as well as into strategies to prevent and treat P. aeruginosa infections and reduce the rate of antibiotic resistance.

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

2001 ◽  
Vol 12 (8) ◽  
pp. 2453-2468 ◽  
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.

scholarly journals The novel Dbl homology/BAR domain protein, MsgA, of Talaromyces marneffei regulates yeast morphogenesis during growth inside host cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Harshini Weerasinghe ◽  
Hayley E. Bugeja ◽  
Alex Andrianopoulos
Keyword(s):  
Pathogenic Fungi ◽  
Host Cells ◽  
Microbial Pathogens ◽  
Mammalian Host ◽  
Host Immune System ◽  
Nucleotide Exchange ◽  
Phagocytic Cells ◽  
Macrophage Infection ◽  
Bar Domain ◽  
Talaromyces Marneffei

AbstractMicrobial pathogens have evolved many strategies to evade recognition by the host immune system, including the use of phagocytic cells as a niche within which to proliferate. Dimorphic pathogenic fungi employ an induced morphogenetic transition, switching from multicellular hyphae to unicellular yeast that are more compatible with intracellular growth. A switch to mammalian host body temperature (37 °C) is a key trigger for the dimorphic switch. This study describes a novel gene, msgA, from the dimorphic fungal pathogen Talaromyces marneffei that controls cell morphology in response to host cues rather than temperature. The msgA gene is upregulated during murine macrophage infection, and deletion results in aberrant yeast morphology solely during growth inside macrophages. MsgA contains a Dbl homology domain, and a Bin, Amphiphysin, Rvs (BAR) domain instead of a Plekstrin homology domain typically associated with guanine nucleotide exchange factors (GEFs). The BAR domain is crucial in maintaining yeast morphology and cellular localisation during infection. The data suggests that MsgA does not act as a canonical GEF during macrophage infection and identifies a temperature independent pathway in T. marneffei that controls intracellular yeast morphogenesis.

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