Comparative sequence analysis of theClostridium difficile toxins A and B

C, von Eichel-Streiber; Laufenberg-Feldmann, R.; Sartingen, S; Schulze, Jürgen P.; Sauerborn, Markus

doi:10.1007/bf00587587

Cited by 149 publications

(105 citation statements)

References 32 publications

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“…Other biochemical aspects, such as the large sizes of toxin subunits, ϳ280 kDa for the protoxin, are not unusual. For example, protein subunits for Clostridium difficile toxins A and B have been calculated to have molecular masses of 308,057 and 269,709 Da, respectively (50). Furthermore, Photorhabdus toxins appear to have similarities to other generic A-B type bacterial toxins that are proteolytically processed to the mature form (51).…”

Section: Ansltalflpqnsrkmentioning

confidence: 99%

Photorhabdus luminescens W-14 Insecticidal Activity Consists of at Least Two Similar but Distinct Proteins

Guo¹,

Fatig²,

Orr³

et al. 1999

Journal of Biological Chemistry

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Both the bacterium Photorhabdus luminescens alone and its symbiotic Photorhabdus-nematode complex are known to be highly pathogenic to insects. The nature of the insecticidal activity of Photorhabdus bacteria was investigated for its potential application as an insect control agent. It was found that in the fermentation broth of P. luminescens strain W-14, at least two proteins, toxin A and toxin B, independently contributed to the oral insecticidal activity against Southern corn rootworm. Purified toxin A and toxin B exhibited single bands on native polyacrylamide gel electrophoresis and two peptides of 208 and 63 kDa on SDS-polyacrylamide gel electrophoresis. The native molecular weight of both the toxin A and toxin B was determined to be approximately 860 kDa, suggesting that they are tetrameric. NH 2 -terminal amino acid sequencing and Western analysis using monospecific antibodies to each toxin demonstrated that the two toxins were distinct but homologous. The oral potency (LD 50 ) of toxin A and toxin B against Southern corn rootworm larvae was determined to be similar to that observed with highly potent Bt toxins against lepidopteran pests. In addition, it was found that the two peptides present in toxin B could be processed in vitro from a 281-kDa protoxin by endogenous P. luminescens proteases. Proteolytic processing was shown to enhance insecticidal activity.

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Section: Ansltalflpqnsrkmentioning

confidence: 99%

Photorhabdus luminescens W-14 Insecticidal Activity Consists of at Least Two Similar but Distinct Proteins

Guo¹,

Fatig²,

Orr³

et al. 1999

Journal of Biological Chemistry

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“…It has been hypothesized that the cluster of 172 hydrophobic, highly conserved amino acids in the middle of the translocation domain (i.e., residues 958-1,130 in TcdA and 956-1,128 in TcdB) comprised some, if not all, of the segments that form the translocation pore (18). Demonstrating this, however, has been challenging, in large part due to difficulties associated with manipulating clostridial toxin genes at the genetic level.…”

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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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“…4,14 The mid portion of the 2 toxins includes hydrophobic regions hypothesized to be associated with membrane insertion and/or translocation. 15,29 Receptor-mediated endocytosis is an essential feature of toxin activity. A number of receptors for TcdA have been proposed or positively identified, including Gala1-3b1-4GlcNAc-R (a-galactosyl or the a-Gal epitope), Galb1-4{Fua1-3}GlcNAc (Lewis X), Galb1-4GlcNAcb1 (Lewis Y), Galb1-14GlcNAcb1-3Galb1-4(Glc) (Lewis I), and sucrose isomaltase.…”

mentioning

confidence: 99%

The Distribution and Density of Clostridium difficile Toxin Receptors on the Intestinal Mucosa of Neonatal Pigs

Keel

Songer

2007

Vet Pathol

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Abstract. Clostridium difficile is an enteric pathogen affecting a variety of mammals, but it has only recently been diagnosed as a cause of neonatal typhlocolitis in pigs. The most important virulence factors of C. difficile are 2 large exotoxins, toxin A (TcdA) and toxin B (TcdB). TcdA is a potent enterotoxin with effects on host tissues that are dependent upon receptor-mediated endocytosis of the intact toxin. TcdB is an effective cytotoxin, but it apparently does not bind receptors on intact mucosal epithelium. TcdB is much less toxic in vivo unless there is underlying damage to the mucosa, and it is not essential for the virulence of C. difficile. One hypothesis to explain the resistance of most species as neonates (e.g., humans and hamsters) is that they may lack significant numbers of TcdA receptors. The susceptibility of neonatal pigs suggests cells of the gastrointestinal mucosa express sufficient numbers of toxin receptors for lesion development. Immunohistochemical (IHC) assays documented specific binding of TcdA, but not TcdB, to the epithelium of the small and large intestine. The carbohydrate Gala1-3b1-4GlcNAc-R has been described as an important receptor for TcdA. However, IHC indicated a distribution on cell surfaces much different from that of TcdA binding, suggesting a specific interaction of toxin with an alternative receptor.

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Comparative sequence analysis of theClostridium difficile toxins A and B

Cited by 149 publications

References 32 publications

Photorhabdus luminescens W-14 Insecticidal Activity Consists of at Least Two Similar but Distinct Proteins

Photorhabdus luminescens W-14 Insecticidal Activity Consists of at Least Two Similar but Distinct Proteins

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

The Distribution and Density of Clostridium difficile Toxin Receptors on the Intestinal Mucosa of Neonatal Pigs

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