The Identification and Purification of Multiple Forms of o-Haemolysin (o-Toxin) of Clostridium perfringens Type A

Smyth, C. J.

doi:10.1099/00221287-87-2-219

Cited by 30 publications

(6 citation statements)

References 16 publications

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“…2), although it had no thiol-activated properties. The amount of cholesterol needed to inhibit the V. metchnikovii cytolysin was about 100 times that generally used to inhibit thiol-activated cytolysin (9,27,34,36,43). Similarly, Gray and Kreger (15) have found that V. vulnificus cytolysin is inhibited by a large amount of cholesterol, but Miyoshi et al (28) have reported that V. vulnificus cytolysin is inhibited by cholesterol at a concentration similar to that which inhibits thiol-activated cytolysin.…”

Section: Discussionmentioning

confidence: 99%

Purification and characterization of Vibrio metschnikovii cytolysin

1988

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An extracellular cytolysin produced by Vibrio metschnikovii was purified by acid precipitation, phenyl-Sepharose CL-4B chromatography, and rechromatography on a phenyl-Sepharose CL-4B column and high-performance liquid chromatography on a Mono Q (anion-exchange) column. The purified cytolysin had a molecular weight of 50,000 and an isoelectric point of 5.1. It was inactivated by heating at 60°C for 5 min and was inhibited by Zn2+, Cu2+, and high concentrations of cholesterol. Lysis of calf erythrocytes by cytolysin was temperature dependent and occurred only above 18°C. Moreover, no lysis was observed at high concentrations of erythrocytes, suggesting that the cytolysin lyses erythrocytes by a multihit mechanism. This cytolysin had no immunological cross-reactivities with hemolysins from other Vibrio species tested, indicating that it is a new cytolysin. V. metschnikovii cytolysin lysed erythrocytes from several animal species (calf, rabbit, guinea pig, mouse, human, sheep, chicken, and horse) and cultured cells (Vero and Chinese hamster ovary), caused fluid accumulation in the intestines of infant mice, and increased vascular permeability in rabbit skin.

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Section: Discussionmentioning

confidence: 99%

Purification and characterization of Vibrio metschnikovii cytolysin

1988

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“…Approximately 6 ,ug of perfringolysin 0 purified from C. perfringens and E. coli carrying pRTlB were separated on a SDS-10% polyacrylamide gel. Lane 1, Molecular weight markers (66,000, 45,000, 36,000, 29,000, 24,000, 20,100,14,200); lane 2, perfringolysin 0 from C. perfringens; lane 3, pfo-encoded perfringolysin 0.…”

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confidence: 99%

Cloning and expression in Escherichia coli of the perfringolysin O (theta-toxin) gene from Clostridium perfringens and characterization of the gene product

Tweten

1988

Infect Immun

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The gene encoding perfringolysin 0, the thiol-activated hemolysin from Clostridium perfringens (ATCC 13124), was cloned and expressed in Escherichia coli. A gene library of C. perfringens chromosomal DNA was constructed in bacteriophage XEMBL3. A recombinant was identified that produced a hemolysin that was inhibited by cholesterol and was tentatively identified as perfringolysin 0. Subcloning experiments localized the perfringolysin 0 gene (pfo) to a 1.8-kilobase region on the cloned chromosomal fragment. E. coli which carried a plasmid subclone of pfo (pRT1B) expressed perfringolysin 0 and secreted it into the periplasm. The amino-terminal sequence of the pfo gene product was identical with that determined for perfringolysin 0 purified from C. perfringens, indicating that E. coli correctly removed the signal peptide during secretion. Purification of the pfo product was accomplished by high-resolution gel filtration and anion-exchange chromatography. Analysis of the pfo product by sodium dodecyl sulfate gel electrophoresis showed that it comigrated with authentic perfringolysin 0; both had an estimated molecular weight of 54,000. Twodimensional tryptic peptide maps of the pfo product and of authentic perfringolysin 0 purified from C. perfringens were identical. The hemolytic activity of the pfo product was similar to that of authentic perfringolysin 0; one hemolytic unit (HU) of the cloned gene product or authentic perfringolysin 0 corresponded to approximately 1 ng or a hemolytic activity of 106 HU per mg.

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“…Two forms of cereolysin separable by discontinuous gel electrophoresis. (A) 25 ,Ag of cereolysin purified in the absence of dithiothreitol was electrophoresed as described in the text, using photopolymerized gels containing 5% acrylamide. (B) 25 ,ug of the same protein sample was electrophoresed as in (A) except that before electrophoresis the protein was incubated with dithiothreitol (0.05 M) for 10 min at 23 C. If the toxin is purified in the presence of 5 mM dithiothreitol, the gel pattern shown in (B) is obtained without preincubation with dithiothreitol.…”

Section: Resultsmentioning

confidence: 99%

“…These are the use of AH-Sepharose and isoelectric focusing. Isoelectric focusing has been used successfully in the isolation of other bacterial toxins (4) and has been used as the principal resolving step in purifying clostridial 0-toxin, the thiol-activated toxin from Clostridium perfringens (25). Although only small amounts of purified cereolysin are obtained, the procedure should be amenable to scaling up.…”

Section: Resultsmentioning

confidence: 99%

“…Recently, a number of these thiol-activated toxins have been shown to possess microheterogeneity. Multiple forms of streptolysin 0 (26), thuringiolysin (21), and clostridial 6-toxin (19,25) have been revealed by isoelectric focusing. In fact, multiple forms of many different bacte-rial toxins have been revealed by isoelectric focusing (4), and, as with the thiol-activated toxins, the cause(s) of the heterogeneity is not clear.…”

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confidence: 99%

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Purification of cereolysin and the electrophoretic separation of the active (reduced) and inactive (oxidized) forms of the purified toxin

Cowell¹,

Grushoff-Kosyk²,

Bernheimer³

1976

Infect Immun

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Cereolysin was purified to apparent homogeneity by using ammonium sulfate fractionation, hydrophobic chromatography with AH-Sepharose, isoelectric focusing, and gel filtration. The active form of the toxin had an isoelectric point of 6.6, and the molecular weight of the protein was about 55,500 as judged by sodium dodecyl sulfate-gel electrophoresis, gel filtration, and gel electrophoresis using various concentrations of acrylamide. Cereolysin contained two halfcystine residues and was dependent on reducing agents, such as dithiothreitol, for maximal hemolytic activity and charge homogeneity. By using discontinuous acrylamide electrophoresis, two forms of the toxin could be observed: oxidized and reduced. If the toxin was purified in the absence of dithiothreitol, partial spontaneous oxidation resulted in the formation of an oxidized form of the toxin. Relative to the reduced form, the oxidized form moved slightly closer to the anode in gel electrophoresis at pH 9.0. If the toxin was purified in the presence of 5 mM dithiothreitol or ifthe spontaneously oxidized toxin was preincubated with dithiothreitol, only the reduced form of the protein was observed. When the logarithms of their relative mobilities were plotted against the concentration of acrylamide in the gels, the slopes for the reduced and oxidized forms were identical. This indicates that the two forms are identical in size and are separable because of different charges. The reduced protein could be inhibited by Nethylmaleimide,5,, and p-hydroxymercuribenzoate. Inhibition by the latter two sulfhydryl reagents could be completely reversed by dithiothreitol. The reversibly oxidized form of the toxin did not appear to be inhibited by N-ethylmaleimide and apparently was either unable to bind to or had a decreased affinity for the erythrocyte membrane.

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The Identification and Purification of Multiple Forms of o-Haemolysin (o-Toxin) of Clostridium perfringens Type A

Cited by 30 publications

References 16 publications

Purification and characterization of Vibrio metschnikovii cytolysin

Purification and characterization of Vibrio metschnikovii cytolysin

Cloning and expression in Escherichia coli of the perfringolysin O (theta-toxin) gene from Clostridium perfringens and characterization of the gene product

Purification of cereolysin and the electrophoretic separation of the active (reduced) and inactive (oxidized) forms of the purified toxin

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