Effects of temperature, pH and detergents on the molecular conformation of the enterotoxin of Clostridium perfringens

Salinovich, Olivia; Mattice, Wayne L.; Blakeney, Ernest W.

doi:10.1016/0167-4838(82)90408-3

Cited by 12 publications

(9 citation statements)

References 21 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: 80%

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: 80%

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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“…4, the peak channel of C. perfringens spores before the treatment was found to be different from that after the treatment, indicating that the heat treatment at 60°C for 30 min destroyed the antigenicity of CPE exposed on the surface of C. perfringens spores. This is supported by the previous report [17] that CPE is a heat-labile protein.…”

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confidence: 79%

Flow Cytometric Analysis for Enterotoxin Exposed on Clostridium Perfringens Spores.

Kusunoki

Piyankarage

et al. 1998

The Journal of Veterinary Medical Science

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ABSTRACT. Flow cytometric method (FCM) with fluorescent-labeled anti-CPE antibody was applied to develop a rapid, specific, and convenient method to detect enterotoxin (CPE) exposed on the surface of spores of Clostridium perfringens. The results obtained indicate that FCM can specifically detect CPE exposed on C. perfringens spores for a short time. Thus, FCM is found to be a rapid, specific, and convenient assay method for detection of CPE exposed on C. perfringens spores, suggesting that it will be hopefully useful to diagnose food poisoning caused by C. perfringens. -KEY WORDS: Clostridium perfringens, enterotoxin, FCM.J. Vet. Med. Sci. 60(12): 1357-1359, 1998 Rabbit antiserum was purified by gel filtration on Sephacryl S-300 (Pharmacia, Uppsala, Sweden). Purified antibody was labeled by the methods of Williams and Chase [24]. Thirty µg of fluorescein isothiocyanate, isomer I (FITC, Wako Pure Chemicals, Osaka, Japan) was added to 1 mg of antibody and incubated at 25°C for 1 hr. Then the mixture was applied to gel filtration on Sephadex G-25 (Pharmacia, Uppsala, Sweden). Further purification of FITC-labeled antibody was carried out by ion-exchange chromatography on DEAE-cellulose (DE-52, Whatman Paper Ltd, Maidstone, England).Heat treatment of formalin-treated C. perfringens spores was carried out by incubating at 60°C for 30 min. The spores (10 8 ) were also treated with 200 µl of rabbit anti-CPE serum by incubating at 37°C for 10 min. Binding of FITC-labeled anti-CPE antibody (40 µl) to either formalintreated vegetative cells or spores (10 6 /ml) was done by incubating at 37°C for 10 min. After these treatment, the spores were washed in PBS by centrifugtlior at 10,000 × g for 10 min.For flow cytometric assay, test samples were prepared as follows: 0.4 ml of formalin-treated vegetative cells or spores (10 8 /ml) was mixed with 50 µl of FITC-labeled purified rabbit anti-CPE antibody for 10 min at 37°C. To study the specificity of FITC-labeled anti-CPE antibody, CPE exposed on C. perfringens spores was treated with unlabeled rabbit anti-CPE antibody prior to FITC-* CORRESPONDENCE TO: Dr. UEMURA, T., Laboratory of Veterinary Public Health, Department of Veterinary Science, College of Agriculture, Osaka Prefecture University, 1-1 Gakuen-cho, Sakai, Osaka 599-8531, Japan.Clostridium perfringens is a common food bacterium distributed widely in human foods, especially meat and poultry products [20]. Type A strains of C. perfringens produce an enterotoxin (CPE) which is the causative factor of human food poisoning [19]. CPE is synthesized during sporulation of C. perfringens vegetative cells and the lysis of sporulated cells liberates CPE into the intestinal tract [7]. Since CPE involves in food poisoning outbreaks caused by C. perfringens, several methods for detection of CPE have been developed over the past few decades [5,12,18,21]. Although enzyme-linked immunosorbent assay (ELISA) is one of the common methods to detect CPE at the present time, it is not useful to analyze CPE exposed on the surface of spores of ...

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“…the 36000-M, aggregate of type A (8-6) is more toxic than the 18000-Mr components. This thesis is further supported by circular dichroism studies [50] which indicated that the native enterotoxin had no alpha helix content at 35°C and pH 6.7. However, on the addition of SDS at 35 "C and pH 6.7 the enterotoxin exhibited 25 -30% helix which compares favourably with the theoretical values calculated in Table 4.…”

Section: Discussionsupporting

confidence: 49%

Purification and properties of an enterotoxin from a coatless spore mutant of Clostridium perfringens type A

Lindsay¹,

Sleigh²,

Ghitgas³

et al. 1985

European Journal of Biochemistry

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A method is described for isolating an enterotoxin from a coatless spore mutant (8-6) of Clostridium perfringens type A. The characteristics of this enterotoxin only slightly resembled those of previously isolated enterotoxins of C. perfringens. The type A (8-6) enterotoxin was found to be composed of two subunits of M , 18000 with isoelectric points of 3.8 and 4.3. The LD5,, for mice was 39 pg/kg with 0.10 pg corresponding to one erythemal unit. The type A (8-6) enterotoxin was inactivated by heating for 10 min at 60°C. The amino acid composition data of type A (8-6) and delta toxins was similar, but type A (8-6) and type A enterotoxins showed less similarity. This lack of similarity between type A and type A (8-6) enterotoxins was confirmed by the failure of anti-sera to type A enterotoxin to neutralize the type A (8-6) enterotoxin, in both the mouse and erythemal tests.Certain strains of Clostridium perfringens type A produce an enterotoxin that is linked to a major cause of mild food poisoning in humans [l]. The enterotoxin is a sporulationspecific gene product, whose production is correlated with late stage I1 to stage I11 of sporulation [2 -51. Several lines of evidence indicate that enterotoxin is possibly excess spore coat protein not incorporated as a structural component of the spore coat, and released from the sporangia only on lysis [6].While a large amount of data is available on the mode of action of enterotoxin [7-91 and more recently on a binding protein [lo] and membrane receptors [ll], information on structure and composition is still subject to some conjecture.The type A enterotoxin isolated by Granum and Skjelkvale [12] is a single polypeptide chain of M , 34000, however, although this molecule has now been fully sequenced [ 13 -151, the question still arises, whether this is the only enterotoxin produced by C. perfringens type A. This concern stems from some unresolved discrepancies in the literature as to the number of subunits, the M , of these subunits, their behaviour under SDS gel electrophoresis and some slight variation in amino acid composition of the enterotoxin when produced under different conditions [16 -231.In our studies on the development of spore heat resistance we have used a strain of C. perfringens type A [24] which produces a spore without a coat, although it produces coat protein(s) and heat-stable spores. Our rationale for this study was that if enterotoxin production is linked to coat protein synthesis, a coatless spore may provide enterotoxin in larger quantities than would normally be obtainable from coated spores. In this paper we report the isolation and characterization of a different enterotoxin from the coatless mutant of C. perfringens type A designated 8-6.Correspondence to J. A. Lindsay, CSIRO Division of Food Research, P. 0. Box 52, North Ryde, Sydney, New South Wales, Australia 21 13Abbreviations. i.p., intraperitoneally ; buffer A, 0.05 M phosphate buffer pH 6.7; SDS, sodium dodecyl sulphate; PAGE, polyacrylamide gel electrophoresis; HPLC, high performanc...

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Effects of temperature, pH and detergents on the molecular conformation of the enterotoxin of Clostridium perfringens

Cited by 12 publications

References 21 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

Flow Cytometric Analysis for Enterotoxin Exposed on Clostridium Perfringens Spores.

Purification and properties of an enterotoxin from a coatless spore mutant of Clostridium perfringens type A

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