Structural determinants for membrane insertion, pore formation and translocation of<i>Clostridium difficile</i>toxin B

Genisyuerek, Selda; Papatheodorou, Panagiotis; Guttenberg, Gregor; Schubert, Rolf; Benz, Roland; Aktories, Klaus

doi:10.1111/j.1365-2958.2011.07549.x

Cited by 97 publications

(139 citation statements)

References 36 publications

Supporting

Mentioning

123

Contrasting

Unclassified

Order By: Relevance

“…Recently, Genisyuerek et al generated a series of internal-, amino-, and carboxyl-terminal truncations of the translocation domain to delineate the determinants of pore formation and translocation for TcdB (21). Consistent with our findings and our model, they concluded that residues 1-1,500 (with a receptorbinding domain) encompassed the translocation machinery enabling functional toxicity.…”

Section: Discussionsupporting

confidence: 85%

“…3B, Top, WT TcdB induced an increase in membrane activity at acidic pH (pH 4.5) with a holding potential of +50 mV (Δψ = +50 mV, cis-positive). The observed large and transient currents were reproducible and similar in character to previous planar lipid bilayer experiments (21). By contrast, despite several attempts (n = 8), with the use of higher concentrations and extended traces, we were unable to detect any channel activity for the L1106K mutant, consistent with an inability to form ion-conductive pores (Fig.…”

Section: High-throughput Mapping Of the Functional Determinants In Thesupporting

confidence: 88%

“…Consistent with our findings and our model, they concluded that residues 1-1,500 (with a receptorbinding domain) encompassed the translocation machinery enabling functional toxicity. Importantly, however, they also showed that a small fragment (i.e., 830-1,025) was able to form channels in planar lipid bilayers (21). Because this fragment only partially overlaps with the residues in our model and excludes the most hydrophobic helices within the delivery domain, this finding would appear to contradict our model; however, it is important to note, as the authors of this study also suggest, that the appearance of ion channel activity in a synthetic lipid bilayer with fragments of a toxin, does not necessarily indicate the existence of the minimal translocation domain.…”

Section: Discussionmentioning

confidence: 99%

“…86 Rb + release assay was performed as previously reported (21). Briefly, CHO-K1 cells were seeded in 96-well plates supplemented with 1 μCi/mL 86 Rb + (PerkinElmer) at a density of 1 × 10 4 cells per well.…”

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%

See 4 more Smart Citations

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.

View full text Add to dashboard Cite

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...

show abstract

Section: Discussionsupporting

confidence: 85%

Section: High-throughput Mapping Of the Functional Determinants In Thesupporting

confidence: 88%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

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.

View full text Add to dashboard Cite

show abstract

Evidence for dual receptor‐binding sites in Clostridium difficile toxin A

Lambert

Baldwin

2016

FEBS Letters

View full text Add to dashboard Cite

TcdA (308 kDa) and TcdB (270 kDa) disrupt the integrity of the intestinal epithelial barrier and provide an environment favorable for Clostridium difficile colonization. Recent evidence suggests that entry of TcdA into cells is mediated by at least two domains. Here, we report the characterization of a second receptor-binding domain (RBD2) for TcdA. While both the isolated combined repetitive oligopeptides (CROPs) and RBD2 fragments are rapidly internalized into cells under physiologic conditions, only the CROPs domain appreciably accumulates at the cell surface. Once internalized, CROPs and RBD2 are trafficked to late endosomal compartments. An internal deletion of RBD2 from TcdA holotoxin ablated toxicity in HT29 cells. These data are consistent with the recently proposed dual receptor model of cellular entry.

show abstract

Receptors and Binding Structures for Clostridium difficile Toxins A and B

Gerhard

2016

Current Topics in Microbiology and Immunology

View full text Add to dashboard Cite

Two characteristics of toxins A and B from C. difficile (TcdA, TcdB) are important for the understanding of the pathogenic effect of these homologous toxins. First, these toxins are huge single-chain but multidomain proteins that display their action intracellularly within the cytosol of host cells. And second, albeit various cell types highly differ in their sensitivity toward these toxins, no toxin-resistant cell type has been described yet. Investigation of receptor-mediated uptake of these toxins is very ambitious. It demands discrimination between cell surface binding, interaction with more than one functional receptor responsible for uptake as well as other functional receptors that recognize bacterial pathogens and are not necessarily related with endocytosis. The current understanding of a complex uptake process is that TcdB interacts with at least two facultative receptors that mediate entry into host cells by redundant endocytotic pathways. Although both homologous toxins do obviously not share the same receptors, this principle of redundant binding domains found for TcdB does also account for TcdA.

show abstract

Structural determinants for membrane insertion, pore formation and translocation ofClostridium difficiletoxin B

Cited by 97 publications

References 36 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

Evidence for dual receptor‐binding sites in Clostridium difficile toxin A

Receptors and Binding Structures for Clostridium difficile Toxins A and B

Contact Info

Product

Resources

About