Cyclophilin A facilitates translocation of the<i>Clostridium botulinum</i>C2 toxin across membranes of acidified endosomes into the cytosol of mammalian cells

Kaiser, Eva; Pust, Sascha; Kroll, Claudia; Barth, Holger

doi:10.1111/j.1462-5822.2009.01291.x

Cited by 73 publications

(126 citation statements)

References 47 publications

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“…This is in agreement with earlier findings on the translocation of the binary toxins iota-toxin and C2 toxin (3,5). Recently, we reported that the membrane translocation of some binary toxins but not of others is facilitated by host cell chaperones and PPIases (7,17,18,22). Therefore, we tested here whether CDT and the closely related iota-toxin require such factors for translocation.…”

Section: Discussionsupporting

confidence: 78%

“…The results corroborate our recent finding that cyclophilins and Hsp90 facilitate the membrane translocation of C2I, the enzyme component of the binary actin-ADP-ribosylating C2 toxin (22). Immunoprecipitation experiments revealed that CypA, the most abundant cyclophilin in the cytosol of mammalian cells and the major target of CsA, interacts with C2I (22). In the present study, we found that CDTa and Ia bound to the immobilized Hsp90 and CypA proteins in vitro, a hint that CypA might be the relevant cyclophilin that interacts with CDT and iota-toxin during cellular uptake too.…”

Section: Vol 79 2011supporting

confidence: 82%

“…Moreover, the translocation of Ia seems to require a membrane potential gradient in addition to the pH gradient (13). We have reported earlier that the membrane translocation of C2, and iota-toxin, is facilitated by the chaperone heat shock protein 90 (Hsp90) (17,18) and more recently that cyclophilin A (CypA), a peptidyl-prolyl cis/trans isomerase (PPIase) (22), is crucial for the translocation of C2 toxin. PPIases catalyze cis-trans isomerization of proline-peptide bonds, often a rate-limiting step during protein refolding (2,9,44,45).…”

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confidence: 99%

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Membrane Translocation of Binary Actin-ADP-Ribosylating Toxins from Clostridium difficile and Clostridium perfringens Is Facilitated by Cyclophilin A and Hsp90

et al. 2011

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Some hypervirulent strains of Clostridium difficile produce the binary actin-ADP-ribosylating toxin C. difficile transferase (CDT) in addition to Rho-glucosylating toxins A and B. It has been suggested that the presence of CDT increases the severity of C. difficile-associated diseases, including pseudomembranous colitis. CDT contains a binding and translocation component, CDTb, that mediates the transport of the separate enzyme component CDTa into the cytosol of target cells, where CDTa modifies actin. Here we investigated the mechanism of cellular CDT uptake and found that bafilomycin A1 protects cultured epithelial cells from intoxication with CDT, implying that CDTa is translocated from acidified endosomal vesicles into the cytosol. Consistently, CDTa is translocated across the cytoplasmic membranes into the cytosol when cell-bound CDT is exposed to acidic medium. Radicicol and cyclosporine A, inhibitors of the heat shock protein Hsp90 and cyclophilins, respectively, protected cells from intoxication with CDT but not from intoxication with toxins A and B. Moreover, both inhibitors blocked the pH-dependent membrane translocation of CDTa, strongly suggesting that Hsp90 and cyclophilin are crucial for this process. In contrast, the inhibitors did not interfere with the ADP-ribosyltransferase activity, receptor binding, or endocytosis of the toxin. We obtained comparable results with the closely related iota-toxin from Clostridium perfringens. Moreover, CDTa and Ia, the enzyme component of iota-toxin, specifically bound to immobilized Hsp90 and cyclophilin A in vitro. In combination with our recently obtained data on the C2 toxin from C. botulinum, these results imply a common Hsp90/cyclophilin A-dependent translocation mechanism for the family of binary actin-ADP-ribosylating toxins.Clostridium difficile infection causes human diseases ranging from mild diarrhea to severe and potentially life-threatening pseudomembranous colitis. C. difficile-associated diseases occur in patients treated with broad-spectrum antibiotics. Under these conditions, the disturbed gut flora allows germination of C. difficile spores and colonization of the gut by this pathogen. C. difficile produces two exotoxins, toxin A (308 kDa) and toxin B (270 kDa), which are the causative agents of pseudomembranous colitis. The toxin-catalyzed glucosylation of Rho, Rac, and Cdc42 results in the inhibition of GTPase-mediated cell signaling, destruction of the actin cytoskeleton, and cell rounding that are the reasons for loss of integrity of the intestinal wall (for a review, see references 20 and 21).During the last 10 years, hypervirulent C. difficile strains were identified that produce, in addition to toxins A and B, a third exotoxin, the binary C. difficile transferase (CDT). Up to 35% of the strains tested produce CDT, and it has been suggested that the presence of CDT correlates with the severity of C. difficile-associated diseases (12,14,25,27,52). CDT belongs to the family of binary actin-ADP-ribosylating toxins and consists of two nonlink...

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Section: Discussionsupporting

confidence: 78%

Section: Vol 79 2011supporting

confidence: 82%

mentioning

confidence: 99%

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Membrane Translocation of Binary Actin-ADP-Ribosylating Toxins from Clostridium difficile and Clostridium perfringens Is Facilitated by Cyclophilin A and Hsp90

et al. 2011

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“…This ratchet mechanism would thus provide the driving force for CTA1 dislocation. Although a previous report suggested the ATP-dependent dislocation of CTA1 does not require cytosolic factors (10), we and others (35,47) have detected a membrane-associated pool of Hsp90 which may have been purified with the microsomal preparation used in that study. Our work clearly shows that Hsp90 exhibits a high affinity interaction with CTA1, and that the disruption of this interaction inhibits both CTA1 dislocation and CT intoxication in vivo.…”

Section: Role Of Hsp90 In Erad-mediated Dislocationmentioning

confidence: 89%

Hsp90 Is Required for Transfer of the Cholera Toxin A1 Subunit from the Endoplasmic Reticulum to the Cytosol

Taylor

Navarro-Garcı́a

Huerta

et al. 2010

Journal of Biological Chemistry

100

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Cholera toxin (CT) is an AB 5 toxin that moves from the cell surface to the endoplasmic reticulum (ER) by retrograde vesicular transport. In the ER, the catalytic A1 subunit dissociates from the rest of the toxin and enters the cytosol by exploiting the quality control system of ER-associated degradation (ERAD). The driving force for CTA1 dislocation into the cytosol is unknown. Here, we demonstrate that the cytosolic chaperone Hsp90 is required for CTA1 passage into the cytosol. Hsp90 bound to CTA1 in an ATP-dependent manner that was blocked by geldanamycin (GA), an established Hsp90 inhibitor. CT activity against cultured cells and ileal loops was also blocked by GA, as was the ER-to-cytosol export of CTA1. Experiments using RNA interference or N-ethylcarboxamidoadenosine, a drug that inhibits ER-localized GRP94 but not cytosolic Hsp90, confirmed that the inhibitory effects of GA resulted specifically from the loss of Hsp90 activity. This work establishes a functional role for Hsp90 in the ERAD-mediated dislocation of CTA1. Cholera toxin (CT)4 is one of the main virulence factors produced by Vibrio cholerae (1, 2). It is an AB-type protein toxin that contains separate catalytic and cell-binding subunits. The catalytic A subunit is initially synthesized as a 27 kDa protein, which undergoes proteolytic nicking to generate a disulfidelinked CTA1/CTA2 heterodimer. The ADP-ribosyltransferase activity of CT resides in the 22 kDa CTA1 polypeptide, while the 5 kDa CTA2 polypeptide maintains numerous non-covalent interactions with the B subunit and thereby links the enzymatic A1 moiety to the cell-binding B moiety. The CTB subunit, built from 11 kDa monomers, is a homopentameric ring-like structure that binds to GM1 gangliosides on the plasma membrane of a target cell.CT travels as an intact holotoxin from the cell surface to the ER (3). Environmental conditions in the ER facilitate reduction of the CTA1/CTA2 disulfide bond and dissociation of CTA1 from CTA2/CTB 5 . This process occurs at the resident redox state of the ER and involves the action of protein-disulfide isomerase (PDI), an ER-localized oxidoreductase (4 -8). Unfolding of the dissociated CTA1 subunit allows it to move into the cytosol through one or more protein-conducting channels in the ER membrane (9 -11). Cytosolic CTA1 then refolds into an active conformation and modifies its Gs␣ target.ER-associated degradation (ERAD), a host quality control mechanism, is responsible for the ER-to-cytosol dislocation of CTA1 (12)(13)(14). A variety of ER-localized chaperones, lectins, and oxidoreductases function in ERAD (15-17). These proteins recognize features that are present in misfolded proteins such as surface-exposed hydrophobic residues or improper patterns of N-linked glycosylation. When a misfolded protein is identified by the ERAD system, it is exported to the cytosol through Sec61 and/or Derlin-1 protein-conducting channels. Dislocated ERAD substrates are usually appended with polyubiquitin chains that serve as a molecular tag for degradation by the 26 S...

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“…To control for this possibility, we directly monitored the ER-to-cytosol translocation of transfected CTA1 in the parental TZM, TZM 1-2, and TZM 5-1 cell lines. Transfected, radiolabeled cells were partitioned into separate membrane and cytosolic fractions according to established protocols (26,29,34,45). Material immunoprecipitated from each fraction with an anti-CTA antibody was then resolved by SDS-PAGE and quantified using PhosphorImager analysis.…”

Section: Pdi-mediated Disassembly Of the Ct Holotoxin Does Notmentioning

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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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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Cyclophilin A facilitates translocation of theClostridium botulinumC2 toxin across membranes of acidified endosomes into the cytosol of mammalian cells

Cited by 73 publications

References 47 publications

Membrane Translocation of Binary Actin-ADP-Ribosylating Toxins from Clostridium difficile and Clostridium perfringens Is Facilitated by Cyclophilin A and Hsp90

Membrane Translocation of Binary Actin-ADP-Ribosylating Toxins from Clostridium difficile and Clostridium perfringens Is Facilitated by Cyclophilin A and Hsp90

Hsp90 Is Required for Transfer of the Cholera Toxin A1 Subunit from the Endoplasmic Reticulum to the Cytosol

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

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