Cholera Toxin: An Intracellular Journey into the Cytosol by Way of the Endoplasmic Reticulum

Wernick, Naomi L. B.; Chinnapen, Daniel J.‐F.; Cho, Jin Ah; Lencer, Wayne I.

doi:10.3390/toxins2030310

Cited by 187 publications

(208 citation statements)

References 83 publications

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“…To reach its cytosolic Gs␣ target, CT moves from the cell surface to the endoplasmic reticulum (ER) by retrograde vesicular traffic (4). A C-terminal KDEL sequence in the CTA2 subunit is thought to target and/or retain CT in the ER (4,5).…”

Section: Cholera Toxin (Ct)mentioning

confidence: 99%

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Protein-disulfide Isomerase Displaces the Cholera Toxin A1 Subunit from the Holotoxin without Unfolding the A1 Subunit

Taylor

Banerjee

Ray

et al. 2011

Journal of Biological Chemistry

127

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Protein-disulfide isomerase (PDI) has been proposed to exhibit an "unfoldase" activity against the catalytic A1 subunit of cholera toxin (CT). Unfolding of the CTA1 subunit is thought to displace it from the CT holotoxin and to prepare it for translocation to the cytosol. To date, the unfoldase activity of PDI has not been demonstrated for any substrate other than CTA1. An alternative explanation for the putative unfoldase activity of PDI has been suggested by recent structural studies demonstrating that CTA1 will unfold spontaneously upon its separation from the holotoxin at physiological temperature. Thus, PDI may simply dislodge CTA1 from the CT holotoxin without unfolding the CTA1 subunit. To evaluate the role of PDI in CT disassembly and CTA1 unfolding, we utilized a real-time assay to monitor the PDI-mediated separation of CTA1 from the CT holotoxin and directly examined the impact of PDI binding on CTA1 structure by isotope-edited Fourier transform infrared spectroscopy. Our collective data demonstrate that PDI is required for disassembly of the CT holotoxin but does not unfold the CTA1 subunit, thus uncovering a new mechanism for CTA1 dissociation from its holotoxin. Cholera toxin (CT)2 is an AB 5 protein toxin that consists of a catalytic A moiety and a cell-binding B moiety (1, 2). The B subunit is pentameric ring-like structure that adheres to GM1 gangliosides on the plasma membrane of a target cell. The A subunit is initially synthesized as a 26 kDa protein that undergoes proteolytic nicking to generate a disulfide-linked A1/A2 heterodimer. The 21 kDa CTA1 polypeptide is an ADP-ribosyltransferase that modifies and activates Gs␣ in the host cell cytosol. CTA1 can be divided into three subdomains: the A1 1 subdomain contains the catalytic core of the toxin; the A1 2 subdomain is a short extended linker that connects the A1 1 and A1 3 subdomains; and the A1 3 subdomain is a globular structure with many hydrophobic residues as well as a cysteine residue involved with the single disulfide bridge between CTA1 and CTA2 (3). The 5 kDa CTA2 polypeptide maintains numerous non-covalent interactions with the central pore of the B pentamer and thereby anchors CTA1 to CTB 5 . A ribbon diagram of the CT holotoxin which highlights the subdomain structure of CTA1 is provided in supplemental Fig. S1.To reach its cytosolic Gs␣ target, CT moves from the cell surface to the endoplasmic reticulum (ER) by retrograde vesicular traffic (4). A C-terminal KDEL sequence in the CTA2 subunit is thought to target and/or retain CT in the ER (4, 5). Conditions in the ER lead to reductive cleavage of the CTA1/CTA2 disulfide bond and chaperone-assisted dissociation of CTA1 from CTA2/CTB 5 (6 -9). Unfolding of the free A1 subunit then activates the quality control system of ER-associated degradation (ERAD), thereby promoting CTA1 translocation to the cytosol (10, 11). Most exported ERAD substrates are degraded by the ubiquitin-proteasome system, but it was hypothesized that CTA1 and the A chains of other ER-translocating toxins avoid t...

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Section: Cholera Toxin (Ct)mentioning

confidence: 99%

“…A C-terminal KDEL sequence in the CTA2 subunit is thought to target and/or retain CT in the ER (4,5). Conditions in the ER lead to reductive cleavage of the CTA1/CTA2 disulfide bond and chaperone-assisted dissociation of CTA1 from CTA2/CTB 5 (6 -9).…”

Section: Cholera Toxin (Ct)mentioning

confidence: 99%

Protein-disulfide Isomerase Displaces the Cholera Toxin A1 Subunit from the Holotoxin without Unfolding the A1 Subunit

Taylor

Banerjee

Ray

et al. 2011

Journal of Biological Chemistry

127

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“…The toxin is composed of the catalytically active A (CTA) subunit associated with five CTB subunits that bind to surface GM 1 gangliosides, facilitating toxin endocytosis (3). In addition to enterotoxigenic activity, CT has also been demonstrated to have immunomodulatory effects (4).…”

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confidence: 99%

Mechanisms of Inflammasome Activation by Vibrio cholerae Secreted Toxins Vary with Strain Biotype

et al. 2015

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Activation of inflammasomes is an important aspect of innate immune responses to bacterial infection. Recent studies have linked Vibrio cholerae secreted toxins to inflammasome activation by using murine macrophages. To increase relevance to human infection, studies of inflammasome-dependent cytokine secretion were conducted with the human THP-1 monocytic cell line and corroborated in primary human peripheral blood mononuclear cells (PBMCs). Both El Tor and classical strains of V. cholerae activated ASC (apoptosis-associated speck-like protein-containing a CARD domain)-dependent release of interleukin-1␤ (IL-1␤) when cultured with human THP-1 cells, but the pattern of induction was distinct, depending on the repertoire of toxins the strains produced. El Tor biotype strains induced release of IL-1␤ dependent on NOD-like receptor family pyrin domain-containing 3 (NLRP3) and ASC due to the secreted pore-forming toxin hemolysin. Unlike in studies with mouse macrophages, the MARTX toxin did not contribute to IL-1␤ release from human monocytic cells. Classical biotype strains, which do not produce either hemolysin or the MARTX toxin, activated low-level IL-1␤ release that was induced by cholera toxin (CT) and dependent on ASC but independent of NLRP3 and pyroptosis. El Tor strains likewise showed increased IL-1␤ production dependent on CT when the hemolysin gene was deleted. In contrast to studies with murine macrophages, this phenotype was dependent on a catalytically active CT A subunit capable of inducing production of cyclic AMP and not on the B subunit. These studies demonstrate that the induction of the inflammasome in human THP-1 monocytes and in PBMCs by V. cholerae varies with the biotype and is mediated by both NLRP3-dependent and -independent pathways. V ibrio cholerae is the causative agent of the diarrheal disease cholera. The O1 serogroup, which is most associated with disease, is divided into two biotypes, classical and El Tor. Classical strains were responsible for the six cholera pandemics that occurred during the 19th century and the first half of the 20th century and are noted for their severe clinical presentation. In 1961, the El Tor strains emerged as the cause of the ongoing seventh pandemic, completely displacing the classical strains from the environment and as a cause of cholera in humans (1). These strains colonize the intestine more effectively and spread between hosts more efficiently but cause fewer deaths and significantly more asymptomatic colonization (2).The primary virulence factor for pandemic cholera strains is cholera toxin (CT), an ADP-ribosylating toxin that disables the G␣ s subunit, resulting in increased cyclic AMP (cAMP) production and secretion of fluid from enterocytes due to the opening of chloride channels. The toxin is composed of the catalytically active A (CTA) subunit associated with five CTB subunits that bind to surface GM 1 gangliosides, facilitating toxin endocytosis (3). In addition to enterotoxigenic activity, CT has also been demonstrated to have immunomodulat...

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“…Toxins, mimicking unfolded proteins, would be transported to the cytosol as part of the cell's quality control system. Evidence that ricin, shiga-like toxin, cytolethal distending toxins, and cholera toxin interact with the ERAD pathway already exists (51)(52)(53)(54). A review of the mitigator list found no clear evidence that this pathway was essential for PE-based immunotoxins.…”

Section: Discussionmentioning

confidence: 99%

Whole-genome RNAi screen highlights components of the endoplasmic reticulum/Golgi as a source of resistance to immunotoxin-mediated cytotoxicity

Pasetto

Antignani

Ormanoglu

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Immunotoxins (antibody–toxin fusion proteins) target surface antigens on cancer cells and kill these cells via toxin-mediated inhibition of protein synthesis. To identify genes controlling this process, an RNAi whole-genome screen (∼22,000 genes at three siRNAs per gene) was conducted via monitoring the cytotoxicity of the mesothelin-directed immunotoxin SS1P. SS1P, a Pseudomonas exotoxin-based immunotoxin, was chosen because it is now in clinical trials and has produced objective tumor regressions in patients. High and low concentrations of SS1P were chosen to allow for the identification of both mitigators and sensitizers. As expected, silencing known essential genes in the immunotoxin pathway, such as mesothelin, furin, KDEL receptor 2, or members of the diphthamide pathway, protected cells. Of greater interest was the observation that many RNAi targets increased immunotoxin sensitivity, indicating that these gene products normally contribute to inefficiencies in the killing pathway. Of the top sensitizers, many genes encode proteins that locate to either the endoplasmic reticulum (ER) or Golgi and are annotated as part of the secretory system. Genes related to the ER-associated degradation system were not among high-ranking mitigator or sensitizer candidates. However, the p97 inhibitor eeyarestatin 1 enhanced immunotoxin killing. Our results highlight potential targets for chemical intervention that could increase immunotoxin killing of cancer cells and enhance our understanding of toxin trafficking.

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Cholera Toxin: An Intracellular Journey into the Cytosol by Way of the Endoplasmic Reticulum

Cited by 187 publications

References 83 publications

Protein-disulfide Isomerase Displaces the Cholera Toxin A1 Subunit from the Holotoxin without Unfolding the A1 Subunit

Protein-disulfide Isomerase Displaces the Cholera Toxin A1 Subunit from the Holotoxin without Unfolding the A1 Subunit

Mechanisms of Inflammasome Activation by Vibrio cholerae Secreted Toxins Vary with Strain Biotype

Whole-genome RNAi screen highlights components of the endoplasmic reticulum/Golgi as a source of resistance to immunotoxin-mediated cytotoxicity

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