Structural Basis for the Function of Clostridium difficile Toxin B

Reinert, D.J.; Jank, Thomas; Aktories, Klaus; Schulz, Georg E.

doi:10.1016/j.jmb.2005.06.071

Cited by 139 publications

(180 citation statements)

References 46 publications

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“…C. difficile toxin B and a related lethal toxin from Clostridium sordellii were reported to associate with anionic lipids through their N-terminal domains. This feature provides the toxins with the lipid membrane environment in which their enzymatic reaction against geranylgeranylated Rho GTPases is facilitated (23,24). Therefore, we examined whether the C1 domain, like the N-terminal domains of the clostridial toxins, has the ability to localize to the lipid membrane (Fig.…”

Section: Resultsmentioning

confidence: 99%

Crystal structures reveal a thiol protease-like catalytic triad in the C-terminal region ofPasteurella multocidatoxin

Kitadokoro

Kamitani²,

Miyazawa³

et al. 2007

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

134

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Pasteurella multocida toxin (PMT), one of the virulence factors produced by the bacteria, exerts its toxicity by up-regulating various signaling cascades downstream of the heterotrimeric GTPases Gq and G12/13 in an unknown fashion. Here, we present the crystal structure of the C-terminal region (residues 575-1,285) of PMT, which carries an intracellularly active moiety. The overall structure of C-terminal region of PMT displays a Trojan horse-like shape, composed of three domains with a ''feet''-,''body''-, and ''head''-type arrangement, which were designated C1, C2, and C3 from the N to the C terminus, respectively. The C1 domain, showing marked similarity in steric structure to the N-terminal domain of Clostridium difficile toxin B, was found to lead the toxin molecule to the plasma membrane. The C3 domain possesses the Cys-HisAsp catalytic triad that is organized only when the Cys is released from a disulfide bond. The steric alignment of the triad corresponded well to that of papain or other enzymes carrying CysHis-Asp. PMT toxicities on target cells were completely abrogated when one of the amino acids constituting the triad was mutated. Our results indicate that PMT is an enzyme toxin carrying the cysteine protease-like catalytic triad dependent on the redox state and functions on the cytoplasmic face of the plasma membrane of target cells.crystallography ͉ cysteine protease ͉ membrane targeting

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Section: Resultsmentioning

confidence: 99%

Crystal structures reveal a thiol protease-like catalytic triad in the C-terminal region ofPasteurella multocidatoxin

Kitadokoro

Kamitani²,

Miyazawa³

et al. 2007

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

134

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“…To address the issue of GT-independent effects, we used recombinant TcdA (rTcdA) (4, 10) and generated two mutant toxins by site-directed mutagenesis of the expression vector. Two highly conserved motifs were chosen for mutation, namely, tryptophan-101 and the DXD motif at positions 285 to 287 (3,17). Analogous mutations of TcdB resulted in reductions of the in vitro GT activity, by factors of 1,000 and 5,000, respectively (5,6,16).…”

Section: Fig 1 (A)mentioning

confidence: 99%

Application of Mutated Clostridium difficile Toxin A for Determination of Glucosyltransferase-Dependent Effects

et al. 2006

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Mutation of tryptophan-101 in Clostridium difficile toxin A, a 308-kDa glucosyltransferase, resulted in a 50-fold-reduced cytopathic activity in cell culture experiments. The mutant toxin A was characterized and applied to distinguish between glucosyltransferase-dependent and -independent effects with respect to RhoB up-regulation as a cellular stress response.Clostridium difficile toxins A and B (TcdA and TcdB, respectively) are the major pathogenicity factors that are causative for antibiotic-associated pseudomembranous colitis (19). Several reports of the in vivo effects of TcdA in animal models reflect efforts to understand the cellular mechanism leading to clinical symptoms as well as to the release of mediators that are involved in the inflammatory process (2,13,15,18,20). The inherent glucosyltransferase

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“…Like many other A-B toxins that mediate their own delivery into cells, high-resolution structures of the enzymic A domains (11)(12)(13) and the receptor-binding portion of the B domains of glucosylating toxin family members are known (14,15), whereas the structure and mechanism of the pore-forming translocation domain remains poorly characterized. These interconnected processes have been proposed to be mediated by the central ∼1,000-aa D domain (i.e., amino acids 801-1,850); however, with the absence of any structural information for this domain in either the prepore or pore state, no framework exists for resolving the functional determinants for this large domain that govern pore formation and translocation.…”

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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Structural Basis for the Function of Clostridium difficile Toxin B

Cited by 139 publications

References 46 publications

Crystal structures reveal a thiol protease-like catalytic triad in the C-terminal region ofPasteurella multocidatoxin

Crystal structures reveal a thiol protease-like catalytic triad in the C-terminal region ofPasteurella multocidatoxin

Application of Mutated Clostridium difficile Toxin A for Determination of Glucosyltransferase-Dependent Effects

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

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