Molecular cloning of the 3' half of the Clostridium perfringens enterotoxin gene and demonstration that this region encodes receptor-binding activity

Hanna, Philip C.; Wnek, Andrew P.; McClane, Bruce A.

doi:10.1128/jb.171.12.6815-6820.1989

Cited by 63 publications

(45 citation statements)

References 33 publications

(24 reference statements)

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“…Structure-function analyses have identified three major functional regions of the CPE protein: an N-terminal cytotoxicity core sequence extending from residue G47 to I51 (22,44), a putative transmembrane stem domain including residues V81 to I106 (44), and a C-terminal binding domain consisting of residues S290 to F319 (14)(15)(16)(17)22). Our current results demonstrate that rCPE 168-319 and rCPE D48A variants bind to the intestinal epithelium, as evident from their positive immunostaining with anti-CPE antibodies.…”

Section: Discussionmentioning

confidence: 99%

“…For example, a synthetic peptide corresponding to the 30 Cterminal amino acids was shown to exhibit strong competitive binding activity against native CPE. In addition, deleting even the five C-terminal amino acids from native CPE was found to completely ablate toxin binding and cytotoxic activities (14)(15)(16)(17)22). However, despite possessing the ability to bind to CPE receptors, the C-terminal half of CPE itself cannot kill cultured cells, which indicates that sequences in the N-terminal half of CPE are required for cytotoxicity (17,18).…”

Section: Clostridium Perfringens Enterotoxin (Cpe) Is Produced By ϳ1mentioning

confidence: 99%

“…In addition, deleting even the five C-terminal amino acids from native CPE was found to completely ablate toxin binding and cytotoxic activities (14)(15)(16)(17)22). However, despite possessing the ability to bind to CPE receptors, the C-terminal half of CPE itself cannot kill cultured cells, which indicates that sequences in the N-terminal half of CPE are required for cytotoxicity (17,18). Deletion and sitedirected mutagenesis approaches have identified amino acids G47 to I51 in the N terminus of CPE as being important for CPE oligomerization and cytotoxicity (21,22,38,44).…”

Section: Clostridium Perfringens Enterotoxin (Cpe) Is Produced By ϳ1mentioning

confidence: 99%

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Noncytotoxic Clostridium perfringens Enterotoxin (CPE) Variants Localize CPE Intestinal Binding and Demonstrate a Relationship between CPE-Induced Cytotoxicity and Enterotoxicity

et al. 2008

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Clostridium perfringens enterotoxin (CPE) causes the symptoms of a very common food poisoning. To assess whether CPE-induced cytotoxicity is necessary for enterotoxicity, a rabbit ileal loop model was used to compare the in vivo effects of native CPE or recombinant CPE (rCPE), both of which are cytotoxic, with those of the noncytotoxic rCPE variants rCPE D48A and rCPE . Both CPE and rCPE elicited significant fluid accumulation in rabbit ileal loops, along with severe mucosal damage that starts at villus tips and then progressively affects the entire villus, including necrosis of epithelium and lamina propria, villus blunting and fusion, and transmural edema and hemorrhage. Similar treatment of ileal loops with either of the noncytotoxic rCPE variants produced no visible histologic damage or fluid transport changes. Immunohistochemistry revealed strong CPE or rCPE 168-319 binding to villus tips, which correlated with the abundant presence of claudin-4, a known CPE receptor, in this villus region. These results support (i) cytotoxicity being necessary for CPE-induced enterotoxicity, (ii) the CPE sensitivity of villus tips being at least partially attributable to the abundant presence of receptors in this villus region, and (iii) claudin-4 being an important intestinal receptor for CPE. Finally, rCPE 168-319 was able to partially inhibit CPEinduced histologic damage, suggesting that noncytotoxic rCPE variants might be useful for protecting against some intestinal effects of CPE.Clostridium perfringens enterotoxin (CPE) is produced by ϳ1 to 5% of all isolates of the gram-positive spore-forming anaerobic pathogen C. perfringens (25). CPE, a single 35-kDa polypeptide of 319 amino acids (6), is responsible for the gastrointestinal symptoms of C. perfringens type A food poisoning (25,39), which is the second most common food-borne illness in the United States and the United Kingdom (36). In addition, CPE-producing C. perfringens isolates are increasingly recognized as a cause of antibiotic-associated or sporadic diarrhea (2,3,27).Studies of CPE effects in experimental animals identified the ileum as the most sensitive region of the small intestine (31, 41). CPE treatment inhibits ileal fluid and electrolyte absorption (30, 31). However, with time, CPE-treated ileum develops gross fluid/electrolyte secretion and histologic damage that includes desquamation of the ileal epithelium, prominent villus blunting, and enterocyte plasma membrane blebbing (32,33,41). Recently, similar histopathologic changes were observed following ex vivo CPE treatment of human ileal tissues, along with increased transepithelial resistance and water permeability (9).In vitro cell culture models have provided considerable information about the cellular and molecular actions of CPE (12,13,24,26,28,34,43). This enterotoxin first binds to certain members of the claudin family of tight junction proteins (19,20,29), which results in formation of an sodium dodecyl sulfate (SDS)-sensitive "small" complex of ϳ90 kDa (48) that contains CPE and one or mor...

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Section: Discussionmentioning

confidence: 99%

Section: Clostridium Perfringens Enterotoxin (Cpe) Is Produced By ϳ1mentioning

confidence: 99%

Section: Clostridium Perfringens Enterotoxin (Cpe) Is Produced By ϳ1mentioning

confidence: 99%

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Noncytotoxic Clostridium perfringens Enterotoxin (CPE) Variants Localize CPE Intestinal Binding and Demonstrate a Relationship between CPE-Induced Cytotoxicity and Enterotoxicity

et al. 2008

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“…Furthermore, those structural analyses, coupled with mutagenesis studies (56)(57)(58)(59)(60)(61), indicated that CPE contains a C-terminal domain that binds to claudin receptors on host cells and an N-terminal domain, consisting of two halves, that is critical for pore formation by mediating oligomerization and membrane insertion.…”

Section: Toxins That Can Be Either Chromosomally or Plasmid Encodedmentioning

confidence: 99%

Toxin Plasmids of Clostridium perfringens

Adams

Bannam

et al. 2013

Microbiol Mol Biol Rev

225

282

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SUMMARY In both humans and animals, Clostridium perfringens is an important cause of histotoxic infections and diseases originating in the intestines, such as enteritis and enterotoxemia. The virulence of this Gram-positive, anaerobic bacterium is heavily dependent upon its prolific toxin-producing ability. Many of the ∼16 toxins produced by C. perfringens are encoded by large plasmids that range in size from ∼45 kb to ∼140 kb. These plasmid-encoded toxins are often closely associated with mobile elements. A C. perfringens strain can carry up to three different toxin plasmids, with a single plasmid carrying up to three distinct toxin genes. Molecular Koch's postulate analyses have established the importance of several plasmid-encoded toxins when C. perfringens disease strains cause enteritis or enterotoxemias. Many toxin plasmids are closely related, suggesting a common evolutionary origin. In particular, most toxin plasmids and some antibiotic resistance plasmids of C. perfringens share an ∼35-kb region containing a Tn 916 -related conjugation locus named tcp (transfer of clostridial plasmids). This tcp locus can mediate highly efficient conjugative transfer of these toxin or resistance plasmids. For example, conjugative transfer of a toxin plasmid from an infecting strain to C. perfringens normal intestinal flora strains may help to amplify and prolong an infection. Therefore, the presence of toxin genes on conjugative plasmids, particularly in association with insertion sequences that may mobilize these toxin genes, likely provides C. perfringens with considerable virulence plasticity and adaptability when it causes diseases originating in the gastrointestinal tract.

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“…Binding activity has been mapped to the extreme C terminus, based upon the binding ability of recombinant CPE (rCPE) fragments containing Cterminal sequences (8)(9)(10)(11) and the binding deficiencies of rCPE fragments lacking 5, 10, or 30 amino acids from the C terminus (17). In contrast, rCPE fragments lacking 36 or 44 amino acids at the N terminus were found to be approximately twofold more cytotoxic than full-length rCPE, indicating that sequences in the N terminus of CPE are mildly inhibitory for cytotoxicity (17).…”

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confidence: 99%

Identification of a Prepore Large-Complex Stage in the Mechanism of Action of Clostridium perfringens Enterotoxin

2007

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Clostridium perfringens enterotoxin (CPE) is the etiological agent of the third most common food-borne illness in the United States. The enteropathogenic effects of CPE result from formation of large CPEcontaining complexes in eukaryotic cell membranes. Formation of these ϳ155-and ϳ200-kDa complexes coincides with plasma membrane permeability changes in eukaryotic cells, causing a Ca 2؉ influx that drives cell death pathways. CPE contains a stretch of amino acids (residues 81 to 106) that alternates markedly in side chain polarity (a pattern shared by the transmembrane domains of the ␤-barrel pore-forming toxin family). The goal of this study, therefore, was to investigate whether this CPE region is involved in pore formation. Complete deletion of the CPE region from 81 to 106 produced a CPE variant that was noncytotoxic for Caco-2 cells and was unable to form CPE pores. However, this variant maintained the ability to form the ϳ155-kDa large complex. This large complex appears to be a prepore present on the plasma membrane surface since it showed greater susceptibility to proteases, increased complex instability, and a higher degree of dissociation from membranes compared to the large complex formed by recombinant CPE. When a D48A mutation was engineered into this prepore-forming CPE variant, the resultant variant was unable to form any prepore ϳ155-kDa large complex. Collectively these findings reveal a new step in CPE action, whereby receptor binding is followed by formation of a prepore large complex, which then inserts into membranes to form a pore.

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Molecular cloning of the 3' half of the Clostridium perfringens enterotoxin gene and demonstration that this region encodes receptor-binding activity

Cited by 63 publications

References 33 publications

Noncytotoxic Clostridium perfringens Enterotoxin (CPE) Variants Localize CPE Intestinal Binding and Demonstrate a Relationship between CPE-Induced Cytotoxicity and Enterotoxicity

Noncytotoxic Clostridium perfringens Enterotoxin (CPE) Variants Localize CPE Intestinal Binding and Demonstrate a Relationship between CPE-Induced Cytotoxicity and Enterotoxicity

Toxin Plasmids of Clostridium perfringens

Identification of a Prepore Large-Complex Stage in the Mechanism of Action of Clostridium perfringens Enterotoxin

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