Clostridium botulinum C2 toxin is internalized by clathrin- and Rho-dependent mechanisms

Pust, Sascha; Barth, Holger; Sandvig, Kirsten

doi:10.1111/j.1462-5822.2010.01512.x

Cited by 42 publications

(37 citation statements)

References 59 publications

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“…Interestingly, C2IN-Stv13 showed partial co-localization with a transferrin-Alexa 647 conjugate, a marker for clathrin-mediated endocytosis (Wittrup and Belting 2009). This finding is consistent with a recent study by Pust and co-workers showing that C2 toxin is internalized by clathrin-and Rho-dependent mechanisms (Pust et al 2010). As expected, C2IN-Stv13 uptake is only marginal in the absence of C2IIa which is in line with the results obtained by cell fractionation and may result from nonspecific pinocytosis.…”

Section: Uptake Of C2in-stv13 Into the Cytosol Of Cancer Cellssupporting

confidence: 94%

“…After assembly of C2IIa with the C2I moiety, the C2IIa/C2I complex binds to its cell surface receptor, an asparagine-linked carbohydrate structure found in all eukaryotic cell types tested (Eckhardt et al 2000;Kaiser et al 2006). This results in receptor-mediated endocytosis of the toxin, which has recently been shown to be clathrin and Rho dependent and to involve PI3K-Akt signaling (Simpson 1989;Nagahama et al 2009;Pust et al 2010). Owing to the acidification of the endosomal lumen, C2IIa undergoes a conformational change and is inserted into the endosomal membrane forming a pore.…”

Section: Introductionmentioning

confidence: 97%

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Internalization of biotinylated compounds into cancer cells is promoted by a molecular Trojan horse based upon core streptavidin and clostridial C2 toxin

Fahrer

Funk

Lillich

et al. 2010

Naunyn-Schmied Arch Pharmacol

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The C2 toxin produced by Clostridium botulinum is a binary AB-type exotoxin composed of the enzyme subunit C2I and the binding/translocation moiety C2II. After proteolytic activation, C2IIa mediates the subsequent internalization of C2I into the cytosol of mammalian target cells. The N-terminal domain of C2I (C2IN) is necessary for C2IIa-dependent uptake, but lacks the enzyme domain that is responsible for cytotoxicity. In the present study, we generated a delivery system building on C2IN and a truncated core streptavidin (Stv13) with enhanced solubility for the C2IIa-dependent internalization of biotinylated cargo molecules into mammalian cells. C2IN-Stv13 fusion protein expressed in Escherichia coli was obtained in high yields and purity. The affinity-purified protein formed tetramers and a defined higher order species in solution as shown by gel filtration and retained its biotin-binding properties, however with an obvious reduction in affinity. Uptake of C2IN-Stv13 into the cytosol of HeLa and other cancer cell lines was observed by immunoblot analysis, which was corroborated by confocal microscopy. In addition, the fusion protein was not cytotoxic and did not inhibit cell proliferation as determined by MTS assay. Finally, we demonstrated the C2IN-Stv13/C2IIa-mediated uptake of biocytin-Alexa 488 as cargo into HeLa cells, underscoring the functionality of the generated transport system.

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Section: Uptake Of C2in-stv13 Into the Cytosol Of Cancer Cellssupporting

confidence: 94%

Section: Introductionmentioning

confidence: 97%

Internalization of biotinylated compounds into cancer cells is promoted by a molecular Trojan horse based upon core streptavidin and clostridial C2 toxin

Fahrer

Funk

Lillich

et al. 2010

Naunyn-Schmied Arch Pharmacol

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“…C2I binds either to cell-bound C2IIa or to soluble C2IIa, and then the preformed C2IIa/ C2I complex can bind to the receptor what might enhance C2 toxin efficiency (Kaiser et al 2006). In any case, the receptor-bound C2IIa/C2I toxin complexes are internalized by receptor-mediated endocytosis (Pust et al 2010), and after transport by the vesicular protein traffic system, C2I translocates from acidified endosomal vesicles into the cytosol (see Fig. 1).…”

Section: Binary Exotoxins and Their Uptake Into Mammalian Cellsmentioning

confidence: 99%

“…However, there is increasing evidence that membrane translocation of other toxins, such as diphtheria or anthrax toxins, is facilitated by cytosolic host cell factors. First, Lemichez and co-workers showed in 1997 that a host cell protein (β-COP) is involved in the cellular uptake of diphtheria toxin (Lemichez et al 1997), and in 2003, Ratts and co-workers demonstrated that Hsp90 and thioredoxin reductase facilitate membrane translocation of this toxin from endosomal vesicles (Ratts et al 2003(Ratts et al , 2005. More recently, Tamayo et al reported that β-COP facilitates translocation of anthrax lethal factor across vesicular membranes in vitro (Tamayo et al 2008).…”

Section: Introductionmentioning

confidence: 96%

Exploring the role of host cell chaperones/PPIases during cellular up-take of bacterial ADP-ribosylating toxins as basis for novel pharmacological strategies to protect mammalian cells against these virulence factors

Barth

2010

Naunyn-Schmied Arch Pharmacol

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Bacterial exotoxins exploit protein transport pathways of their mammalian target cells to deliver their enzymatic active moieties into the cytosol. There, they modify their specific substrate molecules resulting in cell damage and the clinical symptoms characteristic for each individual toxin. We have investigated the cellular uptake of the binary actin ADP-ribosylating C2 toxin from Clostridium botulinum and the binary lethal toxin from Bacillus anthracis, a metalloprotease. Both toxins are composed of a binding/translocation component and a separate enzyme component. During cellular uptake, the binding/translocation components form pores in membranes of acidified endosomes, and the enzyme components translocate as unfolded proteins through the pores into the cytosol. We found by using specific pharmacological inhibitors that the host cell chaperone Hsp90 and the peptidyl-prolyl cis/trans isomerase cyclophilin A are crucial for membrane translocation of the enzyme component of the C2 toxin but not of the lethal toxin, although the structures of the binding/translocation components and the overall uptake mechanisms of both toxins are widely comparable. In conclusion, the new findings imply that Hsp90 and cyclophilin function selectively in promoting translocation of certain bacterial toxins depending on the enzyme domains of the individual toxins. The targeted pharmacological inhibition of individual host cell chaperones/PPIases prevents uptake of certain bacterial exotoxins into the cytosol of mammalian cells and thus protects cells from intoxication. Such substances could represent attractive lead substances for development of novel therapeutics to prevent toxic effects during infection with toxin-producing bacteria.

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“…The binding component is proteolytically activated and forms heptamers, which interact with the enzymatic component (8, 10). After binding of the toxin to a cell surface receptor, the toxin complex is endocytosed (11,12). At low pH of endosomal compartments, the binding component inserts into the membrane of endosomes and forms pores, which allow the translocation of the enzymatic component into the cytosol (10, 13).…”

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confidence: 99%

Cholesterol- and Sphingolipid-rich Microdomains Are Essential for Microtubule-based Membrane Protrusions Induced by Clostridium difficile Transferase (CDT)

Schwan

Nölke

Kruppke

et al. 2011

Journal of Biological Chemistry

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Clostridium difficile toxin (CDT) is a binary actin-ADP-ribosylating toxin that causes depolymerization of the actin cytoskeleton and formation of microtubule-based membrane protrusions, which are suggested to be involved in enhanced bacterial adhesion and colonization of hypervirulent C. difficile strains. Here, we studied the involvement of membrane lipid components of human colon adenocarcinoma (Caco-2) cells in formation of membrane protrusions. Depletion of cholesterol by methyl-␤-cyclodextrin inhibited protrusion formation in a concentration-dependent manner but had no major effect on the toxin-catalyzed modification of actin in target cells. Repletion of cholesterol reconstituted formation of protrusions and increased velocity and total amount of protrusion formation. Methyl-␤-cyclodextrin had no effect on the CDT-induced changes in the dynamics of microtubules. Formation of membrane protrusions was also inhibited by the cholesterol-binding polyene antibiotic nystatin. Degradation or inhibition of synthesis of sphingolipids by sphingomyelinase and myriocin, respectively, blocked CDT-induced protrusion formation. Benzyl alcohol, which increases membrane fluidity, prevented protrusion formation. CDT-induced membrane protrusions were stained by flotillin-2 and by the fluorescent-labeled lipid raft marker cholera toxin subunit B, which selectively interacts with GM1 ganglioside mainly located in lipid microdomains. The data suggest that formation and especially the initiation of CDTinduced microtubule-based membrane protrusions depend on cholesterol-and sphingolipid-rich lipid microdomains.Clostridium difficile causes antibiotic-associated diarrhea and pseudomembranous colitis (1). Both diseases depend on the production of toxins. Major virulence factors are the glycosylating C. difficile toxins A and B, which inactivate Rho GTPases (2-5). Especially hypervirulent strains additionally produce C. difficile transferase (CDT) 3 (6). CDT belongs to the family of actin-ADP-ribosylating toxins like Clostridium perfringens iota toxin and Clostridium botulinum C2 toxin (7-9). These toxins are binary in structure and consist of an enzymatic component, which possesses ADP-ribosyltransferase activity, and a separated binding/translocation component. The binding component is proteolytically activated and forms heptamers, which interact with the enzymatic component (8, 10). After binding of the toxin to a cell surface receptor, the toxin complex is endocytosed (11,12). At low pH of endosomal compartments, the binding component inserts into the membrane of endosomes and forms pores, which allow the translocation of the enzymatic component into the cytosol (10, 13). Here, the toxin ADP-ribosylates actin at arginine 177 (14). Modification at this site inhibits actin polymerization and causes destruction of the actin cytoskeleton.Recently, we reported that following ADP-ribosylation of actin, CDT induces the formation of microtubule-based protrusions in epithelial cells (15). These protrusions are in general Ͻ1 m in diameter ...

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Clostridium botulinum C2 toxin is internalized by clathrin- and Rho-dependent mechanisms

Cited by 42 publications

References 59 publications

Internalization of biotinylated compounds into cancer cells is promoted by a molecular Trojan horse based upon core streptavidin and clostridial C2 toxin

Internalization of biotinylated compounds into cancer cells is promoted by a molecular Trojan horse based upon core streptavidin and clostridial C2 toxin

Exploring the role of host cell chaperones/PPIases during cellular up-take of bacterial ADP-ribosylating toxins as basis for novel pharmacological strategies to protect mammalian cells against these virulence factors

Cholesterol- and Sphingolipid-rich Microdomains Are Essential for Microtubule-based Membrane Protrusions Induced by Clostridium difficile Transferase (CDT)

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