Intoxication of zebrafish and mammalian cells by cholera toxin depends on the flotillin/reggie proteins but not Derlin-1 or -2
David E. Saslowsky, … , Barry H. Paw, Wayne I. Lencer
David E. Saslowsky, … , Barry H. Paw, Wayne I. Lencer
Published November 1, 2010
Citation Information: J Clin Invest. 2010;120(12):4399-4409. https://doi.org/10.1172/JCI42958.
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Research Article Cell biology

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

Abstract

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.

Authors

David E. Saslowsky, Jin Ah Cho, Himani Chinnapen, Ramiro H. Massol, Daniel J.-F. Chinnapen, Jessica S. Wagner, Heidi E. De Luca, Wendy Kam, Barry H. Paw, Wayne I. Lencer

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Figure 2

Zebrafish embryos model the pathways co-opted by CT to induce toxicity in mammalian cells.

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Zebrafish embryos model the pathways co-opted by CT to induce toxicity i...
(A) Protein lysates were prepared from pools of five 16-hpf embryos incubated 1 hour with no toxin (control), 5 nM WT CT, or 5 nM G33D and analyzed for CTB and β-actin by immunoblot. Purified CTB was loaded as standard (std). (B) Total gangliosides from 16-hpf zebrafish embryos (embryo extract) or bovine brain gangliosides (BBG) were analyzed by TLC along with GM1 authentic standard (left panel). The GM1 standard and embryo extract were combined and also analyzed (lane 4). Filled arrowheads indicate resorcinol-staining GM1 band in GM1 standard and BBG. A portion of these extracts were subjected to CTB overlay immunoblot analysis (right panel). (C) Embryos (16-hpf) from 3 parental zebrafish strains (AB, Tü, and WIK) were incubated with embryo water alone (control) or with the indicated toxins at 5 nM for 48 hours. Only WT CT produced in V. cholerae or E. coli (CT and rCT, respectively) induced toxicity (asterisks). Toxin schematics at left indicate mutations in the G33D B-subunit and R192G A-subunit. n ≥ 50 for each intoxicated strain; images are representative of at least 95% of the population. (D) Embryos were intoxicated as indicated (5 nM) or mock treated (no toxin) as in C and assayed at 48 hpf for total cAMP content by ELISA. Results from pools of 10 embryos/condition from 3 independent experiments are represented as mean ± SD. (E) Embryos (16-hpf) were treated with vehicle (control; 0.01% DMSO, n = 85) or 10 μM forskolin (n = 57) and imaged at 62 hpf (images representative of 100% of each population).