Interaction of Clostridium perfringens θ-haemolysin, a contaminant of commercial phospholipase C, with erythrocyte ghost membranes and lipid dispersions. A morphological study

Smyth, C. J.; Freer, John H.; Arbuthnott, J. P.

doi:10.1016/0005-2736(75)90216-3

Cited by 50 publications

(19 citation statements)

References 42 publications

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“…These latter formations could represent cellular attempts to recycle membrane fragments translocated to the cell surface during degranulation [36]. Alternatively, similar TAC-toxin-induced changes in erythrocyte membranes have been described [37,38] and have been suggested to consist of toxin-cholesterol aggregates [39][40][41][42] that contribute to cell lysis via a colloid-osmotic mechanism [43]. It is of interest that early toxin-induced ultrastructural changes included the coalescence of the multi-lobed nucleus with homogenous redistribution of the nuclear contents, and that disruption of the nuclear membrane occurred in the absence of plasma membrane lysis (Fig.…”

Section: Discussionmentioning

confidence: 91%

Clostridium perfringens invasiveness is enhanced by effects of theta toxin upon PMNL structure and function: The role of leukocytotoxicity and expression of CD11/CD18 adherence glycoprotein

Bryant¹

1993

FEMS Immunology and Medical Microbiology

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Clostridium perfringens infections are characterized by the lack of an inflammatory response at the site of infection and rapidly progressive margins of tissue necrosis. Studies presented here investigated the role of theta toxin from C. perfringens in the pathophysiology of these events. Mice passively immunized with neutralizing monoclonal antibody against theta toxin and challenged with an LD100 of log phase C. perfringens had significantly less mortality than untreated controls. Intramuscular injection of killed, washed C. perfringens in mice induced a massive time-dependent influx of polymorphonuclear leukocytes (PMNL) into tissue; injection of either viable, washed C. perfringens or killed organisms plus theta toxin dramatically attenuated PMNL influx although PMNL accumulated in adjacent vessels. The anti-inflammatory effects could not be attributed to an absence of chemoattractants since C. perfringens proteins had chemotactic factor activity, and killed bacilli generated serum-derived chemotactic factors. Scanning and transmission electron microscopy demonstrated the dramatic leukocidal effects of high doses of theta toxin on PMNL. In contrast, sublethal concentrations of theta toxin primed PMNL chemiluminescence, disrupted PMNL cytoskeletal actin polymerization/disassembly, and stimulated functional upregulation of CD11b/CD18 adherence glycoprotein. In summary, these results demonstrate that theta toxin is an important virulence factor in C. perfringens infection. In a concentration-dependent fashion, theta toxin contributes to the pathogenesis of clostridial gangrene by direct destruction of host inflammatory cells and tissues, and by promoting dysregulated PMNL/endothelial cell adhesive interactions.

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Section: Discussionmentioning

confidence: 91%

Clostridium perfringens invasiveness is enhanced by effects of theta toxin upon PMNL structure and function: The role of leukocytotoxicity and expression of CD11/CD18 adherence glycoprotein

Bryant¹

1993

FEMS Immunology and Medical Microbiology

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“…Recently, Smyth et al (27) demonstrated that the membrane lesions produced by commercial phospholipase C (9) were due to contamination by the Clostridium perfringens 0-hemolysin. The 0hemolysin is also an oxygen-labile toxin, and Smyth and co-workers found that it produced ringand arc-shaped structures in erythrocyte membranes, lipid dispersions containing cholesterol, and cholesterol dispersions very similar to the structures described here.…”

Section: Discussionmentioning

confidence: 99%

Effect of streptolysin O on erythrocyte membranes, liposomes, and lipid dispersions. A protein-cholesterol interaction.

Duncan¹,

Schlegel²

1975

The Journal of Cell Biology

135

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The effect of the bacterial cytolytic toxin, streptolysin O (SLO), on rabbit erythrocyte membranes, liposomes, and lipid dispersions was examined. SLO produced no gross alterations in the major erythrocyte membrane proteins or lipids. However, when erythrocytes were treated with SLO and examined by electron microscopy, rings and "C"-shaped structures were observed in the cell membrane. The rings had an electron-dense center, 24 nm in diameter, and the overall diameter of the structure was 38 nm. Ring formation also occurred when erythrocyte membranes were fixed with glutaraldehyde and OsO4 before the addition of toxin. In contrast, rings were not seen when erythrocytes were treated with toxin at 0~indicating that adsorption of SLO to the membrane is not sufficient for ring formation since toxin is known to bind to erythrocytes at that temperature. The ring structures were present on lecithin-cholesterol-dicetylphosphate liposomes after SLO treatment, but there was no release of the trapped, internal markers, K2CrO4 or glucose. The crucial role of cholesterol in the formation of rings and C's was demonstrated by the fact that these structures were present in toxin-treated cholesterol dispersions, but not in lecithin-dicetylphosphate dispersions nor in the SLO preparations alone. The importance of cholesterol was also shown by the finding that no rings were present in membranes or cholesterol dispersions which had been treated with digitonin before SLO was added. Although the rings do not appear to be "holes" in the membrane, a model is proposed which suggests that cholesterol molecules are sequestered during ring and C-structure formation, and that this process plays a role in SLO-induced hemolysis.Streptolysin O (SLO) is a bacterial toxin produced by virtually all strains of Streptococcus pyogenes. The toxin is a protein with a molecular weight of approximately 60,000 daltons, and is characteristic of a group of cytolytic toxins known as the oxygen-labile toxins (3). The toxins in this group are produced by several different gram-positive bacteria, and possess a number of common properties: they are activated by SH compounds: they appear to be antigenically related; and their biological activity is completely inhibited by low concentrations of cholesterol and certain related sterols.Hemolysis occurs within minutes after the addi-160 THE JOURNAL OF CELL BIOLOGY 9 VOLUME 67, 1975 9 pages 160-173 tion of SLO to erythrocytes, and toxic effects of SLO on several types of mammalian cells in culture have been demonstrated (13). The speed with which sensitive cells are affected by the toxin suggests that the cell membrane is the primary site of action. Several lines of indirect evidence suggest that membrane cholesterol is the binding site and/or target of SLO action. Only those cells which contain cholesterol in their membranes are susceptible to the toxin, SLO is "inactivated" only by the membrane lipid fraction which contains cholesterol, and the addition of exogenous cholesterol to SLO inhibits toxin action....

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“…However, most previous studies on the biological activities of alpha-toxin have employed either crude commercial preparations or purified preparations for which the criteria for purity have not been exacting. It has been demonstrated that such material is frequently contaminated with up to ten other C. perfringens products, many of which have significant activities with respect to biological membranes (Mollby ef at., 1973;Smyth et al, 1975). For example, Mollby et al (1973; demonstrated that up to 90% of the cytolytic activity of partially purified alpha-toxin detected in assays employing isotonic ceil suspensions is the result of contamination with theta-toxin.…”

Section: Introductionmentioning

confidence: 99%

Phospholipase C and haemolytic activities of Clostridium perfringens alpha‐toxin cloned in Escherichia coli: sequence and homology with a Baciilus cereus phospholipase C

Leslie

Fairweather

Pickard

et al. 1989

Molecular Microbiology

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The Clostridium perfringens alpha-toxin (phospholipase C) gene (cpa) has been cloned and expressed in Escherichia coli. The biological activities of the cloned gene product have been analysed and the complete nucleotide sequence of the cpa gene has been determined. The cloned cpa gene product, which is exported to the periplasm in E. coli, possesses both phospholipase C and haemolytic activities. Haemolysis is not apparent when cell extracts are incubated with isotonic suspensions of sheep erythrocytes, but can be detected and quantified readily when dilutions of the same extracts are placed in wells in sheep-blood agar plates. Like other sequenced clostridial genes, the cpa gene has a high AT content (66.4%), exhibits a strong bias for using codons with A or T in the wobble position, and the 350 base pairs upstream from the gene have a significantly higher AT content (79.5%) than the coding region. The cpa gene encodes a 398 amino acid polypeptide with a deduced molecular weight of 45,481 D. This is very similar to the estimated molecular weight (Mr) of the cpa primary gene product expressed in an in vitro transcription-translation system (Mr 46,000), but larger than the cpa gene product detected in E. coli minicells, E. coli whole cells or in C. perfringens cells (Mr 43,000), suggesting post-translational processing. The 28 N-terminal residues of the deduced alpha-toxin sequence possess the consensus features of a signal peptide and may be removed during secretion. The deduced alpha-toxin sequence shares significant structural homology with the phosphatidylcholine-preferring phospholipase C of Bacillus cereus.

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Interaction of Clostridium perfringens θ-haemolysin, a contaminant of commercial phospholipase C, with erythrocyte ghost membranes and lipid dispersions. A morphological study

Cited by 50 publications

References 42 publications

Clostridium perfringens invasiveness is enhanced by effects of theta toxin upon PMNL structure and function: The role of leukocytotoxicity and expression of CD11/CD18 adherence glycoprotein

Clostridium perfringens invasiveness is enhanced by effects of theta toxin upon PMNL structure and function: The role of leukocytotoxicity and expression of CD11/CD18 adherence glycoprotein

Effect of streptolysin O on erythrocyte membranes, liposomes, and lipid dispersions. A protein-cholesterol interaction.

Phospholipase C and haemolytic activities of Clostridium perfringens alpha‐toxin cloned in Escherichia coli: sequence and homology with a Baciilus cereus phospholipase C

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