Clostridium perfringens type A enterotoxin forms mepacrine-sensitive pores in pure phospholipid bilayers in the absence of putative receptor proteins

Hardy, Simon P.; Ritchie, C. B; Allen, Marcus; Ashley, Richard H.; Granum, Per Einar

doi:10.1016/s0005-2736(01)00391-1

Cited by 28 publications

(32 citation statements)

References 20 publications

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“…Oligomerization of some ␤-PFTs involves N-terminal sequences that serve as a "latch" domain, linking two neighboring monomers (e.g., Staphylococcus aureus alpha-hemolysin and LukF hemolysin [30,36,[38][39][40]). CPE also has a high ␤-sheet composition (7,32) and a ␤-PFT-like ability to induce membrane permeability changes in sensitive cells (24) and forms pores or channels in artificial membranes (15,37). Our present data appear consistent with the possibility that the CPE N-terminal core sequence identified in this study acts as an N-terminal latch that mediates the protein-protein interactions needed for membrane permeability alterations.…”

Section: Discussionsupporting

confidence: 86%

“…The N-terminal 45-53 region of CPE under examination in this study is required (19) for the plasma membrane permeability alterations responsible for CPE-induced killing of mammalian cells (15,23,24,26,37). Therefore, the ability of each alanine-substituted rCPE variant to induce plasma membrane permeability changes in CaCo-2 cells was assessed in a 86 Rb release assay.…”

Section: Vol 72 2004 Site-directed Mutagenesis Of Cpe's Cytotoxicitmentioning

confidence: 99%

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Fine Mapping of the N-Terminal Cytotoxicity Region of Clostridium perfringens Enterotoxin by Site-Directed Mutagenesis

Smedley

McClane

2004

Infect Immun

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Clostridium perfringens enterotoxin (CPE) has a unique mechanism of action that results in the formation of large, sodium dodecyl sulfate-resistant complexes involving tight junction proteins; those complexes then induce plasma membrane permeability alterations in host intestinal epithelial cells, leading to cell death and epithelial desquamation. Previous deletion and point mutational studies mapped CPE receptor binding activity to the toxin's extreme C terminus. Those earlier analyses also determined that an N-terminal CPE region between residues D45 and G53 is required for large complex formation and cytotoxicity. To more finely map this N-terminal cytotoxicity region, site-directed mutagenesis was performed with recombinant CPE (rCPE). Alanine-scanning mutagenesis produced one rCPE variant, D48A, that failed to form large complexes or induce cytotoxicity, despite having normal ability to bind and form the small complex. Two saturation variants, D48E and D48N, also had a phenotype resembling that of the D48A variant, indicating that both size and charge are important at CPE residue 48. Another alanine substitution rCPE variant, I51A, was highly attenuated for large complex formation and cytotoxicity, but rCPE saturation variants I51L and I51V displayed a normal large complex formation and cytotoxicity phenotype. Collectively, these mutagenesis results identify a core CPE sequence extending from residues G47 to I51 that directly participates in large complex formation and cytotoxicity.

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Section: Discussionsupporting

confidence: 86%

Section: Vol 72 2004 Site-directed Mutagenesis Of Cpe's Cytotoxicitmentioning

confidence: 99%

Fine Mapping of the N-Terminal Cytotoxicity Region of Clostridium perfringens Enterotoxin by Site-Directed Mutagenesis

Smedley

McClane

2004

Infect Immun

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“…Investigation of the ability of purified Nhe to form single channel-like pores in synthetic phosphatidylethanolamine-phosphatidylserine (PE-PS) lipid bilayers was carried out as described previously (Hardy et al, 2001b). Purified Nhe components in 0.1 M NaCl phosphate buffer in a ratio of 1 : 1 : 0.3 NheA : NheB : NheC, using approximately 40 ng NheA, were premixed before addition to the earthed bathing solution.…”

Section: Methodsmentioning

confidence: 99%

Bacillus cereus Nhe is a pore-forming toxin with structural and functional properties similar to the ClyA (HlyE, SheA) family of haemolysins, able to induce osmotic lysis in epithelia

et al. 2008

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The mechanism by which Bacillus cereus causes diarrhoea is unknown. Three putative enterotoxins have been proposed, haemolysin BL (Hbl), cytotoxin K and non-haemolytic enterotoxin (Nhe). Both Hbl and Nhe are three-component cytotoxins and maximal cytotoxicity of Nhe against epithelia is dependent on all three components. However, little is known of the mechanism of cytotoxicity. Markers of plasma membrane disruption, namely propidium iodide uptake, loss of cellular ATP and release of lactate dehydrogenase (LDH), were observed in epithelia exposed to Nhe from culture supernatants of B. cereus, but not in those exposed to supernatants from a mutant strain lacking NheB and NheC. Consistent with an exogenous cause of membrane damage, purified Nhe components combined to form large conductance pores in planar lipid bilayers. The inhibition of LDH release by osmotic protectants and the increase in cell size caused by Nhe indicate that epithelia lyse following osmotic swelling. Nhe and Hbl show sequence homology, and Hbl component B has remarkable structural similarities to cytolysin A (ClyA), with both structures possessing an a-helix bundle and a unique subdomain containing a hydrophobic b-hairpin. Correspondingly, we show that Nhe has haemolytic activity against erythrocytes from a variety of species. We propose that the common structural and functional properties indicate that the Hbl/Nhe and ClyA families of toxins constitute a superfamily of pore-forming cytotoxins.

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“…The CH-1 prepore complex, which forms in both cultured Caco-2 cells and the small intestine, then inserts into membranes by using a ␤-hairpin formed by CPE amino acids 81 to 106 (59,61). This process results in formation of a cation-selective CPE pore that is initially permeable to molecules of Ͻ200 Da (1,(75)(76)(77).…”

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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Clostridium perfringens type A enterotoxin forms mepacrine-sensitive pores in pure phospholipid bilayers in the absence of putative receptor proteins

Cited by 28 publications

References 20 publications

Fine Mapping of the N-Terminal Cytotoxicity Region of Clostridium perfringens Enterotoxin by Site-Directed Mutagenesis

Fine Mapping of the N-Terminal Cytotoxicity Region of Clostridium perfringens Enterotoxin by Site-Directed Mutagenesis

Bacillus cereus Nhe is a pore-forming toxin with structural and functional properties similar to the ClyA (HlyE, SheA) family of haemolysins, able to induce osmotic lysis in epithelia

Toxin Plasmids of Clostridium perfringens

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