Expression of recombinant Clostridium difficile toxin A and B in Bacillus megaterium

Yang, Guang; Zhou, Boping; Wang, Jufang; He, Xiangyun; Sun, Xingmin; Nie, Weijia; Tzipori, Saul; Feng, Hanping

doi:10.1186/1471-2180-8-192

Cited by 114 publications

(130 citation statements)

References 44 publications

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“…Recombinant TcdB wild type was a B. megaterium expression vector pHis1522 encoding the strain VPI10463 obtained from Hangping Feng (University of Maryland, Baltimore). Proteins were expressed and purified as previously described (20).…”

Section: Methodsmentioning

confidence: 99%

“…Demonstrating this, however, has been challenging, in large part due to difficulties associated with manipulating clostridial toxin genes at the genetic level. The recent availability of clones of both TcdA and TcdB in Bacillus megaterium expression plasmids, which enable the high-level production of stably folded toxin, has facilitated research in this direction (19,20); however, studies specifically addressing the structure and function of the translocation domain have been limited to large-fragment deletions to probe function (21,22).In the present study, we set out initially with the goal of identifying the determinants of pore formation and translocation through a comprehensive mutagenesis study using the B. megaterium platform. We discovered very early in this pursuit that Significance Clostridium difficile is the leading cause of antibiotic-associated infection in hospitals worldwide.…”

mentioning

confidence: 99%

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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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Section: Methodsmentioning

confidence: 99%

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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“…49 Briefly, vero cells in 96-well plates were exposed to TcdA/TcdB or mTcd138 at different concentrations. MTT [3-(4,5-dimethylthiazol-2-yl) 2 2,5-diphenyl tetrazolium bromide] assays were used to determine the toxin or mTcd138 cytotoxicity.…”

Section: Cytotoxicity Of Mtcd138 In Cellsmentioning

confidence: 99%

“…49 Recombinant TcdA and TcdB were expressed in B. megaterium and purified as described previously. 49 To generate mTcd138, the DNA sequences from C. difficile VPI 10463, encoding the glucosyltransferase (GTD 1-543 aa) with 2 amino acid mutations (W102A and D288N) and cysteine proteinase (CPD, 543-767) domains of TcdB and receptor binding domain (RBD) of TcdA were bridged with a linker (ggt ggc tct ggt) sequence, synthesized by Geneart (Germany) and cloned between the BsrGI and EagI sites of the vector pHis1525. mTcd138 was expressed in B. megeatarium and purified as described previously.…”

Section: Expression Of Recombinant Fusion Protein Mtcd138 In Bacillusmentioning

confidence: 99%

A chimeric protein comprising the glucosyltransferase and cysteine proteinase domains of toxin B and the receptor binding domain of toxin A induces protective immunity againstClostridium difficileinfection in mice and hamsters

Wang

Yan

Kim

et al. 2015

Human Vaccines & Immunotherapeutics

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“…Recombinant CDTa and CDTb (from C. difficile strain 196) were produced as His-tagged proteins in the Bacillus megaterium expression system as was described previously for the clostridial glycosylating toxins (18,19). All binding components (CDTb and C2II) used in this study were used as protease-activated proteins according to Refs.…”

Section: Methodsmentioning

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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Expression of recombinant Clostridium difficile toxin A and B in Bacillus megaterium

Abstract: Background: Major Clostridium difficile virulence factors are the exotoxins TcdA and TcdB. Due to the large size and poor stability of the proteins, the active recombinant TcdA and TcdB have been difficult to produce.

Cited by 114 publications

References 44 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

A chimeric protein comprising the glucosyltransferase and cysteine proteinase domains of toxin B and the receptor binding domain of toxin A induces protective immunity againstClostridium difficileinfection in mice and hamsters

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

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