Conversion of apical plasma membrane sphingomyelin to ceramide attenuates the intoxication of host cells by cholera toxin

Saslowsky, David E.; Lencer, Wayne I.

doi:10.1111/j.1462-5822.2007.01015.x

Cited by 19 publications

(36 citation statements)

References 63 publications

(93 reference statements)

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“…Once in the cytosol CTA1 recovers the active conformation required for the ADP-ribosylation of its target, the α subunit of the heterotrimeric G protein (Gsα) [28,29] that activates the adenylate cyclase, giving rise to increased levels of cellular cyclic AMP (cAMP) [30]. It is well known that CTB binding and cross-linking of five GM1 molecules serves to promote the function of lipid rafts in toxin trafficking, in fact lowering the expression of GM1 or cholesterol can prevent CT endocytosis [31][32][33]. However, how GM1 undertakes a specific lipid trafficking route has not been completely assessed.…”

Section: Discussionmentioning

confidence: 99%

Cell Propagation of Cholera Toxin CTA ADP-Ribosylating Factor by Exosome Mediated Transfer

Zanetti¹,

Gallina²,

Fabbri³

et al. 2018

Preprint

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Abstract:Here we first report, how cholera toxin (CT) A subunit (CTA), the bacterial enzyme moiety responsible of cell signaling alteration, can take over the exosomal pathway, spread extracellularly and be transmitted in a cell population. A first evidence for long-term transmission of CT toxic effect via extracellular vesicles was obtained in CHO cells. To follow CT intracellular route towards exosome secretion we adopted a strategy apt to convert multivesicular body (MVB) derived exosomes in traceable fluorescent vectors. CT treated Me665 cells, a human melanoma cell line highly expressing caveolin-1 (Cav-1) and GM1, were used to purify and characterize fluorescent

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Section: Discussionmentioning

confidence: 99%

Cell Propagation of Cholera Toxin CTA ADP-Ribosylating Factor by Exosome Mediated Transfer

Zanetti¹,

Gallina²,

Fabbri³

et al. 2018

Preprint

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“…IP of CTB was as described before (13), and some samples were then affinity-purified of biotin conjugates using NeutrAvidinagarose (Thermo Scientific). SDS-PAGE, Western blot analysis, and phosphorimaging was as described (13).…”

Section: Tgn/er Trafficking Assay and Selectivementioning

confidence: 99%

Ganglioside GM1-mediated Transcytosis of Cholera Toxin Bypasses the Retrograde Pathway and Depends on the Structure of the Ceramide Domain

Saslowsky

Welscher

Chinnapen

et al. 2013

Journal of Biological Chemistry

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Background: Mechanisms for intracellular lipid sorting remain poorly understood. Results: Polarized epithelial cells sort ganglioside GM1, the receptor for cholera toxin, into distinct retrograde and transcytotic pathways, provided that GM1 contains ceramide domains with short or unsaturated fatty acid chains. Conclusion: Sphingolipid sorting depends on ceramide structure, implicating a mechanism for lipid sorting by lipid shape. Significance: The results identify a lipid-sorting pathway across epithelial barriers with clinical applications.

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“…In Cos-1 cells, CTB binding and competition were as previously described (8), except that 3 nM CT holotoxin was applied to Cos-1 cells and a 500-fold molar excess of CTB was utilized (1.5 μM). Toxin endocytosis in Cos-1 cells transfected with control and flotillin siRNAs was as previously described (8) with the following modifications: Cos-1 cells grown in one 10-cm 2 well/sample were incubated with 1 nM CT for 75 minutes on ice, washed of excess toxin, and kept on ice or warmed to 37°C for 0, 2, or 10 minutes to promote endocytosis. Three rounds of acid stripping was performed by incubating cells with 2 ml of 100 mM glycine, pH 2.0, for 5 minutes and then with HBSS, pH 7.4, for 5 minutes.…”

Section: Reagents and Antibodies Vibrio Cholerae Ct And Ctb Were Purmentioning

confidence: 99%

“…To determine maximal surface-bound CT, parallel samples were washed only with 4°C HBSS, pH 7.4 (no acid strip; note 7-fold fewer of these samples were loaded in Figure 6B). Cell lysis, immunoprecipitation, and immunoblotting were as previously described (8).…”

Section: Reagents and Antibodies Vibrio Cholerae Ct And Ctb Were Purmentioning

confidence: 99%

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Intoxication of zebrafish and mammalian cells by cholera toxin depends on the flotillin/reggie proteins but not Derlin-1 or -2

Saslowsky¹,

Cho²,

Chinnapen³

et al. 2010

J. Clin. Invest.

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Cholera toxin (CT) causes the massive secretory diarrhea associated with epidemic cholera. To induce disease, CT enters the cytosol of host cells by co-opting a lipid-based sorting pathway from the plasma membrane, through the trans-Golgi network (TGN), and into the endoplasmic reticulum (ER). In the ER, a portion of the toxin is unfolded and retro-translocated to the cytosol. Here, we established zebrafish as a genetic model of intoxication and examined the Derlin and flotillin proteins, which are thought to be usurped by CT for retro-translocation and lipid sorting, respectively. Using antisense morpholino oligomers and siRNA, we found that depletion of Derlin-1, a component of the Hrd-1 retro-translocation complex, was dispensable for CT-induced toxicity. In contrast, the lipid raft-associated proteins flotillin-1 and -2 were required. We found that in mammalian cells, CT intoxication was dependent on the flotillins for trafficking between plasma membrane/endosomes and two pathways into the ER, only one of which appears to intersect the TGN. These results revise current models for CT intoxication and implicate protein scaffolding of lipid rafts in the endosomal sorting of the toxin-GM1 complex. IntroductionCholera toxin (CT) is an AB 5 -subunit toxin responsible for the massive secretory diarrhea seen in epidemic cholera. As for most toxins, CT must gain access to the cytosol of host cells to cause disease. The strategy employed by CT is to bind ganglioside GM1 in the plasma membrane (PM) via the B-subunit (CTB). GM1 carries the toxin retrograde through endosomes, the trans-Golgi network (TGN), and likely all the way into the ER (1, 2). In the ER, a portion of the A-subunit (CTA), the A1-chain, crosses to the cytosol by coopting the machinery that retro-translocates terminally misfolded proteins for degradation by the proteasome (termed ER-associated degradation [ERAD]; refs. 3, 4). The A1-chain refolds in the cytosol and activates adenylate cyclase to increase cAMP. The mechanisms for lipid sorting and ERAD usurped by CT are fundamental to eukaryotic cell biology but remain incompletely understood. To explore how CT exploits these pathways in an unbiased way, we used the zebrafish as a model because it is amenable to genetic screens. Here, we show that CT intoxicates zebrafish embryos by hijacking the same basic mechanisms used in mammalian cells and examine the dependence of CT toxicity on two families of proteins implicated in toxin action: the flotillins and Derlins. These proteins have emerged as important components of lipid-based trafficking and ERAD, respectively.There is evidence that GM1 sorts CT retrograde from PM to ER by association with lipid rafts (2, 5-8). Lipid rafts are cooperative selfassemblies of lipids and proteins that influence various aspects of

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Conversion of apical plasma membrane sphingomyelin to ceramide attenuates the intoxication of host cells by cholera toxin

Cited by 19 publications

References 63 publications

Cell Propagation of Cholera Toxin CTA ADP-Ribosylating Factor by Exosome Mediated Transfer

Cell Propagation of Cholera Toxin CTA ADP-Ribosylating Factor by Exosome Mediated Transfer

Ganglioside GM1-mediated Transcytosis of Cholera Toxin Bypasses the Retrograde Pathway and Depends on the Structure of the Ceramide Domain

Intoxication of zebrafish and mammalian cells by cholera toxin depends on the flotillin/reggie proteins but not Derlin-1 or -2

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