Clostridial Glucosylating Toxins Enter Cells via Clathrin-Mediated Endocytosis

Papatheodorou, Panagiotis; Zamboglou, Constantinos; Genisyuerek, Selda; Guttenberg, Gregor; Aktories, Klaus

doi:10.1371/journal.pone.0010673

Cited by 144 publications

(133 citation statements)

References 51 publications

(55 reference statements)

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“…The series of events leading to the delivery of the A domain into cells begins with toxin binding to an as yet unidentified receptor on target cells via the C-terminal receptor-binding domain (i.e., the B domain), which triggers toxin internalization into acidified vesicles via clathrin-mediated endocytosis (5). In the endosome, cryptic regions from within the large ∼1,000-aa translocation domain emerge and insert into the endosomal membrane, creating a pore that is believed to enable translocation of the N-terminal glucosyltransferase (i.e., the A domain) into the cytosol.…”

mentioning

confidence: 99%

Translocation domain mutations affecting cellular toxicity identify the Clostridium difficile toxin B pore

Zhang

Park

Tam

et al. 2014

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

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Disease associated with Clostridium difficile infection is caused by the actions of the homologous toxins TcdA and TcdB on colonic epithelial cells. Binding to target cells triggers toxin internalization into acidified vesicles, whereupon cryptic segments from within the 1,050-aa translocation domain unfurl and insert into the bounding membrane, creating a transmembrane passageway to the cytosol. Our current understanding of the mechanisms underlying pore formation and the subsequent translocation of the upstream cytotoxic domain to the cytosol is limited by the lack of information available regarding the identity and architecture of the transmembrane pore. Here, through systematic perturbation of conserved sites within predicted membrane-insertion elements of the translocation domain, we uncovered highly sensitive residues-clustered between amino acids 1,035 and 1,107-that when individually mutated, reduced cellular toxicity by as much as >1,000-fold. We demonstrate that defective variants are defined by impaired pore formation in planar lipid bilayers and biological membranes, resulting in an inability to intoxicate cells through either apoptotic or necrotic pathways. These findings along with the unexpected similarities uncovered between the pore-forming "hotspots" of TcdB and the wellcharacterized α-helical diphtheria toxin translocation domain provide insights into the structure and mechanism of formation of the translocation pore for this important class of pathogenic toxins.T he primary virulence determinants of pathogenic Clostridium difficile are two protein toxins, TcdA and TcdB, which are responsible for the symptoms associated with infection, including diarrhea and pseudomembranous colitis (1). TcdA and TcdB are large (i.e., 308 and 270 kDa, respectively) homologous toxins (sharing 48% sequence identity) that appear to intoxicate target cells using a strategy that is similar in principle to that described for a number of smaller A-B toxins, such as anthrax toxin (2) and diphtheria toxin (DT) (3). In addition to a cytotoxic enzymic A domain and receptor-binding B domain responsible for binding and translocating the A domain into cells, TcdA and TcdB are additionally equipped with an internal autoprocessing domain that proteolytically cleaves and releases the N-terminal glucosyltransferase domain in response to intracellular inositol hexakisphosphate (4).The series of events leading to the delivery of the A domain into cells begins with toxin binding to an as yet unidentified receptor on target cells via the C-terminal receptor-binding domain (i.e., the B domain), which triggers toxin internalization into acidified vesicles via clathrin-mediated endocytosis (5). In the endosome, cryptic regions from within the large ∼1,000-aa translocation domain emerge and insert into the endosomal membrane, creating a pore that is believed to enable translocation of the N-terminal glucosyltransferase (i.e., the A domain) into the cytosol. Processed and released A chains enzymatically glucosylate and thereby inactivat...

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mentioning

confidence: 99%

Translocation domain mutations affecting cellular toxicity identify the Clostridium difficile toxin B pore

Zhang

Park

Tam

et al. 2014

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

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“…Initially, the CROPs are thought to bind to some unknown molecules on the cell surface, facilitating toxin entry into cells via receptor-mediated endocytosis (18)(19)(20). Once the endosome is acidified, the toxins undergo a conformational change (21), inserting the transmembrane region into the endosomal membrane and translocating the CPD and GTD into the cytosol (22, 23).…”

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confidence: 99%

Critical Roles of Clostridium difficile Toxin B Enzymatic Activities in Pathogenesis

Shi

Yang

et al. 2015

Infect Immun

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TcdB is one of the key virulence factors of Clostridium difficile that is responsible for causing serious and potentially fatal colitis. The toxin contains at least two enzymatic domains: an effector glucosyltransferase domain for inactivating host Rho GTPases and a cysteine protease domain for the delivery of the effector domain into host cytosol. Here, we describe a novel intrabody approach to examine the role of these enzymes of TcdB in cellular intoxication. By screening a single-domain heavy chain (V H H) library raised against TcdB, we identified two V H H antibodies, 7F and E3, that specifically inhibit TcdB cysteine protease and glucosyltransferase activities, respectively. Cytoplasmic expression of 7F intrabody in Vero cells inhibited TcdB autoprocessing and delayed cellular intoxication, whereas E3 intrabody completely blocked the cytopathic effects of TcdB holotoxin. These data also demonstrate for the first time that toxin autoprocessing occurs after cysteine protease and glucosyltransferase domains translocate into the cytosol of target cells. We further determined the role of the enzymatic activities of TcdB in in vivo toxicity using a sensitive systemic challenge model in mice. Consistent with these in vitro results, a cysteine protease noncleavable mutant, TcdB-L543A, delayed toxicity in mice, whereas glycosyltransferase-deficient TcdB demonstrated no toxicity up to 500-fold of the 50% lethal dose (LD 50 ) when it was injected systemically. Thus, glucosyltransferase but not cysteine protease activity is critical for TcdB-mediated cytopathic effects and TcdB systemic toxicity, highlighting the importance of targeting toxin glucosyltransferase activity for future therapy. C lostridium difficile is an anaerobic Gram-positive bacterial species that can induce serious and potentially fatal inflammatory disease of the colon and is the most prevalent cause of antibioticassociated diarrhea and pseudomembranous colitis in nosocomial settings (1, 2). Disease in patients with C. difficile infection is strongly associated with the two exotoxins, TcdA and TcdB (3). Both toxins are large, homologous single-chain proteins that contain at least four distinct domains (4-6): the N terminus glucosyltransferase domain (GTD), a cysteine protease domain (CPD), a translocation domain (TD), and a C terminus receptor binding domain (RBD; also known as combined repetitive oligopeptides, or CROPs). A recent study suggests that there might also be an additional receptor binding region besides the N-terminal CROP region (7) although the specific region has yet to be identified. Both toxins exert cytopathic effects that include cell rounding after disruption of the actin cytoskeleton and tight junctions in human colonocytes (8, 9). Toxin exposure may also trigger potent cytotoxic and inflammatory effects leading to mucosal cell death, diarrhea, and colitis associated with C. difficile infections (10, 11). TcdB appears to be more clinically relevant for C. difficile virulence as it is invariably associated with clinically isol...

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“…Ia and Ib were purified as described earlier (33). Recombinant CDTa and CDTb (from C. difficile strain 196) were produced and purified as His-tagged proteins in the B. megaterium expression system as described by others for the large clostridial glycosylating toxins (32,55). Labeling of CDTa with Alexa 568-maleimide was performed according to the manufacturer's protocol (Invitrogen, Karlsruhe, Germany).…”

Section: Methodsmentioning

confidence: 99%

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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Clostridial Glucosylating Toxins Enter Cells via Clathrin-Mediated Endocytosis

Cited by 144 publications

References 51 publications

Translocation domain mutations affecting cellular toxicity identify the Clostridium difficile toxin B pore

Translocation domain mutations affecting cellular toxicity identify the Clostridium difficile toxin B pore

Critical Roles of Clostridium difficile Toxin B Enzymatic Activities in Pathogenesis

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

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